Chapter 6 Chapter 6 Chapter 6 Chapter 6 EnzymesEnzymesEnzymesEnzymes

1. An Introduction to Enzymes1. An Introduction to Enzymes1. An Introduction to Enzymes1. An Introduction to Enzymes• Enzymes are catalytically active biological Enzymes are catalytically active biological Enzymes are catalytically active biological Enzymes are catalytically active biological

macromoleculesmacromoleculesmacromoleculesmacromolecules• Enzymes are catalysts of biological systemsEnzymes are catalysts of biological systemsEnzymes are catalysts of biological systemsEnzymes are catalysts of biological systems• Almost every biochemical reaction is catalyzed Almost every biochemical reaction is catalyzed Almost every biochemical reaction is catalyzed Almost every biochemical reaction is catalyzed

by an enzymeby an enzymeby an enzymeby an enzyme• Study of enzymatic processes is the oldest field Study of enzymatic processes is the oldest field Study of enzymatic processes is the oldest field Study of enzymatic processes is the oldest field

of biochemistry, dating back to late 1700sof biochemistry, dating back to late 1700sof biochemistry, dating back to late 1700sof biochemistry, dating back to late 1700s• Study of enzymes has dominated biochemistry Study of enzymes has dominated biochemistry Study of enzymes has dominated biochemistry Study of enzymes has dominated biochemistry

in the past and continues to do soin the past and continues to do soin the past and continues to do soin the past and continues to do so

An Introduction to EnzymesAn Introduction to EnzymesAn Introduction to EnzymesAn Introduction to Enzymes• Most enzymes are globular proteins, however, Most enzymes are globular proteins, however, Most enzymes are globular proteins, however, Most enzymes are globular proteins, however,

some catalytic RNAs (ribozymes) also some catalytic RNAs (ribozymes) also some catalytic RNAs (ribozymes) also some catalytic RNAs (ribozymes) also catalyze reactionscatalyze reactionscatalyze reactionscatalyze reactions

• Many enzymes require nonprotein coenzymes Many enzymes require nonprotein coenzymes Many enzymes require nonprotein coenzymes Many enzymes require nonprotein coenzymes or cofactors for their catalytic activityor cofactors for their catalytic activityor cofactors for their catalytic activityor cofactors for their catalytic activity

• Enzymes are classified according to the type Enzymes are classified according to the type Enzymes are classified according to the type Enzymes are classified according to the type of reactions they catalyzeof reactions they catalyzeof reactions they catalyzeof reactions they catalyze

• All enzymes have formal E.C. numbers and All enzymes have formal E.C. numbers and All enzymes have formal E.C. numbers and All enzymes have formal E.C. numbers and names and most have trivial namesnames and most have trivial namesnames and most have trivial namesnames and most have trivial names

Subdivision of CofactorsSubdivision of CofactorsSubdivision of CofactorsSubdivision of Cofactors• Metals Metals Metals Metals• Small organic molecules (coenzymes) Small organic molecules (coenzymes) Small organic molecules (coenzymes) Small organic molecules (coenzymes)• A coenzyme or metal ion that is very A coenzyme or metal ion that is very A coenzyme or metal ion that is very A coenzyme or metal ion that is very

tightly or even covalently bound to the tightly or even covalently bound to the tightly or even covalently bound to the tightly or even covalently bound to the enzyme protein is called a enzyme protein is called a enzyme protein is called a enzyme protein is called a prosthetic groupprosthetic groupprosthetic groupprosthetic group

• A complete, catalytically active enzyme A complete, catalytically active enzyme A complete, catalytically active enzyme A complete, catalytically active enzyme together with its bound coenzyme and/or together with its bound coenzyme and/or together with its bound coenzyme and/or together with its bound coenzyme and/or metal ions is called a metal ions is called a metal ions is called a metal ions is called a holoenzymeholoenzymeholoenzymeholoenzyme

• The protein part of such an enzyme is The protein part of such an enzyme is The protein part of such an enzyme is The protein part of such an enzyme is called the called the called the called the apoenzymeapoenzymeapoenzymeapoenzyme or or or or apoproteinapoproteinapoproteinapoprotein

Classification of EnzymesClassification of EnzymesClassification of EnzymesClassification of Enzymes• Enzymes are classified according to the type Enzymes are classified according to the type Enzymes are classified according to the type Enzymes are classified according to the type

of reactions they catalyzeof reactions they catalyzeof reactions they catalyzeof reactions they catalyze• Enzymes are divided into six classes, each Enzymes are divided into six classes, each Enzymes are divided into six classes, each Enzymes are divided into six classes, each

with subclasseswith subclasseswith subclasseswith subclasses• Each enzyme is assigned a four-part Each enzyme is assigned a four-part Each enzyme is assigned a four-part Each enzyme is assigned a four-part classification number and a systematic name, classification number and a systematic name, classification number and a systematic name, classification number and a systematic name, which identifies the reaction it catalyzes which identifies the reaction it catalyzes which identifies the reaction it catalyzes which identifies the reaction it catalyzes• Most enzyme have trivial namesMost enzyme have trivial namesMost enzyme have trivial namesMost enzyme have trivial names

Example of Enzyme NamingExample of Enzyme NamingExample of Enzyme NamingExample of Enzyme NamingATP + D-glucose ADP+glucose-6-phosphateATP + D-glucose ADP+glucose-6-phosphateATP + D-glucose ADP+glucose-6-phosphateATP + D-glucose ADP+glucose-6-phosphate• The The The The formal systematic name formal systematic name formal systematic name formal systematic name of the enzyme catalyzing the of the enzyme catalyzing the of the enzyme catalyzing the of the enzyme catalyzing the

reaction is reaction is reaction is reaction is ATP:glucose phosphotransferaseATP:glucose phosphotransferaseATP:glucose phosphotransferaseATP:glucose phosphotransferase• Its Enzyme Commission number (Its Enzyme Commission number (Its Enzyme Commission number (Its Enzyme Commission number (E.C. number) is 2.7.1.1E.C. number) is 2.7.1.1E.C. number) is 2.7.1.1E.C. number) is 2.7.1.1. . . . • The first number (2) denotes the class name (transferase) The first number (2) denotes the class name (transferase) The first number (2) denotes the class name (transferase) The first number (2) denotes the class name (transferase) • The second number (7), the subclass (phosphotransferase)The second number (7), the subclass (phosphotransferase)The second number (7), the subclass (phosphotransferase)The second number (7), the subclass (phosphotransferase)• The third number (1), a phosphotransferase with a hydroxyl The third number (1), a phosphotransferase with a hydroxyl The third number (1), a phosphotransferase with a hydroxyl The third number (1), a phosphotransferase with a hydroxyl

group as acceptor;group as acceptor;group as acceptor;group as acceptor;• The fourth number (1), D-glucose as the phosphoryl group The fourth number (1), D-glucose as the phosphoryl group The fourth number (1), D-glucose as the phosphoryl group The fourth number (1), D-glucose as the phosphoryl group

acceptor. acceptor. acceptor. acceptor. • The The The The trivial name trivial name trivial name trivial name is is is is hexokinasehexokinasehexokinasehexokinase

Enzymes are classified by the reactions they catalyzeEnzymes are classified by the reactions they catalyzeEnzymes are classified by the reactions they catalyzeEnzymes are classified by the reactions they catalyze (www.chem.qmul.ac.uk/iubmb/enzyme) (www.chem.qmul.ac.uk/iubmb/enzyme) (www.chem.qmul.ac.uk/iubmb/enzyme) (www.chem.qmul.ac.uk/iubmb/enzyme)

Characteristics of Enzymes Characteristics of Enzymes Characteristics of Enzymes Characteristics of Enzymes• Higher reaction ratesHigher reaction ratesHigher reaction ratesHigher reaction rates• Greater reaction specificityGreater reaction specificityGreater reaction specificityGreater reaction specificity• Milder reaction conditionsMilder reaction conditionsMilder reaction conditionsMilder reaction conditions• Capacity for regulationCapacity for regulationCapacity for regulationCapacity for regulation• Enzymes affect the rate of a reaction, not Enzymes affect the rate of a reaction, not Enzymes affect the rate of a reaction, not Enzymes affect the rate of a reaction, not

equilibriumequilibriumequilibriumequilibrium• Enzymes lower the activation energyEnzymes lower the activation energyEnzymes lower the activation energyEnzymes lower the activation energy• Enzymes use binding energy to lower the Enzymes use binding energy to lower the Enzymes use binding energy to lower the Enzymes use binding energy to lower the

activation energyactivation energyactivation energyactivation energy• Enzymes are not used up in the reactionEnzymes are not used up in the reactionEnzymes are not used up in the reactionEnzymes are not used up in the reaction

The two most striking The two most striking The two most striking The two most striking characteristics of enzymescharacteristics of enzymescharacteristics of enzymescharacteristics of enzymes

1. 1. 1. 1. Catalytic capacityCatalytic capacityCatalytic capacityCatalytic capacity---Enzymes accelerate reactions by ---Enzymes accelerate reactions by ---Enzymes accelerate reactions by ---Enzymes accelerate reactions by a factor of 10a factor of 10a factor of 10a factor of 105555 to 10 to 10 to 10 to 1017171717. Most reactions in biological . Most reactions in biological . Most reactions in biological . Most reactions in biological systems do not take place at perceptible rates in the systems do not take place at perceptible rates in the systems do not take place at perceptible rates in the systems do not take place at perceptible rates in the absence of enzymes.absence of enzymes.absence of enzymes.absence of enzymes.

2. 2. 2. 2. SpecificitySpecificitySpecificitySpecificity

Specificity of EnzymesSpecificity of EnzymesSpecificity of EnzymesSpecificity of Enzymes• Enzymes are highly specific both in the reactions that Enzymes are highly specific both in the reactions that Enzymes are highly specific both in the reactions that Enzymes are highly specific both in the reactions that

they catalyze and in their choice of reactants, which they catalyze and in their choice of reactants, which they catalyze and in their choice of reactants, which they catalyze and in their choice of reactants, which are called substrates.are called substrates.are called substrates.are called substrates.

• An enzyme usually catalyzes a single chemical reaction An enzyme usually catalyzes a single chemical reaction An enzyme usually catalyzes a single chemical reaction An enzyme usually catalyzes a single chemical reaction or a set of closely related reactions.or a set of closely related reactions.or a set of closely related reactions.or a set of closely related reactions.

• Side reactions leading to the wasteful formation of by-Side reactions leading to the wasteful formation of by-Side reactions leading to the wasteful formation of by-Side reactions leading to the wasteful formation of by-products are rare in enzyme-catalyzed reactions.products are rare in enzyme-catalyzed reactions.products are rare in enzyme-catalyzed reactions.products are rare in enzyme-catalyzed reactions.

• The specificity of an enzyme is due to the precise The specificity of an enzyme is due to the precise The specificity of an enzyme is due to the precise The specificity of an enzyme is due to the precise interaction of the substrate with the enzyme. This interaction of the substrate with the enzyme. This interaction of the substrate with the enzyme. This interaction of the substrate with the enzyme. This precision is a result of the intricate three-dimensional precision is a result of the intricate three-dimensional precision is a result of the intricate three-dimensional precision is a result of the intricate three-dimensional structure of the enzyme protein.structure of the enzyme protein.structure of the enzyme protein.structure of the enzyme protein.

Examples Demonstrating the Specificity of EnzymesExamples Demonstrating the Specificity of EnzymesExamples Demonstrating the Specificity of EnzymesExamples Demonstrating the Specificity of Enzymes

Proteolytic enzymes catalyze proteolysis, the Proteolytic enzymes catalyze proteolysis, the Proteolytic enzymes catalyze proteolysis, the Proteolytic enzymes catalyze proteolysis, the hydrolysis of a peptide bond.hydrolysis of a peptide bond.hydrolysis of a peptide bond.hydrolysis of a peptide bond.

Enzyme SpecificityEnzyme SpecificityEnzyme SpecificityEnzyme Specificity

TrypsinTrypsinTrypsinTrypsin: cleaves on the : cleaves on the : cleaves on the : cleaves on the carboxyl side of arginine carboxyl side of arginine carboxyl side of arginine carboxyl side of arginine and lysine residuesand lysine residuesand lysine residuesand lysine residues

ThrombinThrombinThrombinThrombin: cleaves Arg-Gly : cleaves Arg-Gly : cleaves Arg-Gly : cleaves Arg-Gly bonds in particular bonds in particular bonds in particular bonds in particular sequencessequencessequencessequences

2. Free Energy Is a Useful Thermodynamic 2. Free Energy Is a Useful Thermodynamic 2. Free Energy Is a Useful Thermodynamic 2. Free Energy Is a Useful Thermodynamic Function for Understanding Enzymes Function for Understanding Enzymes Function for Understanding Enzymes Function for Understanding Enzymes

1. The free energy difference between the products 1. The free energy difference between the products 1. The free energy difference between the products 1. The free energy difference between the products and reactants. (It determines whether the and reactants. (It determines whether the and reactants. (It determines whether the and reactants. (It determines whether the reaction will be spontaneous.)reaction will be spontaneous.)reaction will be spontaneous.)reaction will be spontaneous.)

2. The energy required to initiate the conversion of 2. The energy required to initiate the conversion of 2. The energy required to initiate the conversion of 2. The energy required to initiate the conversion of reactants to products. (It determines the rate of reactants to products. (It determines the rate of reactants to products. (It determines the rate of reactants to products. (It determines the rate of the reaction.)the reaction.)the reaction.)the reaction.)

The free energy of the system is plotted against the progress of the reaction S The free energy of the system is plotted against the progress of the reaction S The free energy of the system is plotted against the progress of the reaction S The free energy of the system is plotted against the progress of the reaction S →→→→ P P P P A diagram of this kind is a description of the energy changes during the reaction, A diagram of this kind is a description of the energy changes during the reaction, A diagram of this kind is a description of the energy changes during the reaction, A diagram of this kind is a description of the energy changes during the reaction,

and the horizontal axis (reaction coordinate) reflects the progressive chemical and the horizontal axis (reaction coordinate) reflects the progressive chemical and the horizontal axis (reaction coordinate) reflects the progressive chemical and the horizontal axis (reaction coordinate) reflects the progressive chemical changes (e.g., bond breakage or formation) as S is converted to P. changes (e.g., bond breakage or formation) as S is converted to P. changes (e.g., bond breakage or formation) as S is converted to P. changes (e.g., bond breakage or formation) as S is converted to P.

The activation energies, The activation energies, The activation energies, The activation energies, ΔΔΔΔGGGG˚̊̊̊, for the S , for the S , for the S , for the S →→→→ P and P P and P P and P P and P →→→→ S reactions are indicated. S reactions are indicated. S reactions are indicated. S reactions are indicated. ΔΔΔΔGGGG′′′′˚̊̊̊ is the overall standard free-energy change in the direction S is the overall standard free-energy change in the direction S is the overall standard free-energy change in the direction S is the overall standard free-energy change in the direction S →→→→ P. P. P. P.

△G = G (products) G = G (products) G = G (products) G = G (products) –––– G (substrates) G (substrates) G (substrates) G (substrates)• △G depends only on the free energy of the products and G depends only on the free energy of the products and G depends only on the free energy of the products and G depends only on the free energy of the products and

the free energy of the reactantsthe free energy of the reactantsthe free energy of the reactantsthe free energy of the reactants• △G is independent of the molecular mechanism of the G is independent of the molecular mechanism of the G is independent of the molecular mechanism of the G is independent of the molecular mechanism of the

transformationtransformationtransformationtransformation• △G provides no information about the rate of a reactionG provides no information about the rate of a reactionG provides no information about the rate of a reactionG provides no information about the rate of a reaction

△G <0 --- the reaction can occur spontaneouslyG <0 --- the reaction can occur spontaneouslyG <0 --- the reaction can occur spontaneouslyG <0 --- the reaction can occur spontaneously△G =0 --- the reaction is in equilibriumG =0 --- the reaction is in equilibriumG =0 --- the reaction is in equilibriumG =0 --- the reaction is in equilibrium△G>0 --- the reaction cannot occur spontaneouslyG>0 --- the reaction cannot occur spontaneouslyG>0 --- the reaction cannot occur spontaneouslyG>0 --- the reaction cannot occur spontaneously

The Free-Energy Change Provides Information The Free-Energy Change Provides Information The Free-Energy Change Provides Information The Free-Energy Change Provides Information About the Spontaneity but Not the Rate of a ReactionAbout the Spontaneity but Not the Rate of a ReactionAbout the Spontaneity but Not the Rate of a ReactionAbout the Spontaneity but Not the Rate of a Reaction

• An enzyme cannot alter the laws of An enzyme cannot alter the laws of An enzyme cannot alter the laws of An enzyme cannot alter the laws of thermodynamics and consequently cannot alter thermodynamics and consequently cannot alter thermodynamics and consequently cannot alter thermodynamics and consequently cannot alter the equilibrium of a chemical reaction (the equilibrium of a chemical reaction (the equilibrium of a chemical reaction (the equilibrium of a chemical reaction (△G)G)G)G)

• This inability means that an enzyme accelerates This inability means that an enzyme accelerates This inability means that an enzyme accelerates This inability means that an enzyme accelerates the forward and reverse reactions by precisely the forward and reverse reactions by precisely the forward and reverse reactions by precisely the forward and reverse reactions by precisely the same factorthe same factorthe same factorthe same factor

Enzymes Alter Only the Reaction RateEnzymes Alter Only the Reaction RateEnzymes Alter Only the Reaction RateEnzymes Alter Only the Reaction RateNot the Reaction EquilibriumNot the Reaction EquilibriumNot the Reaction EquilibriumNot the Reaction Equilibrium

Enzymes function to lower the activation energy Enzymes function to lower the activation energy Enzymes function to lower the activation energy Enzymes function to lower the activation energy

• Enzymes accelerate reactions by decreasing Enzymes accelerate reactions by decreasing Enzymes accelerate reactions by decreasing Enzymes accelerate reactions by decreasing GGGG‡‡‡‡, the free energy of activation., the free energy of activation., the free energy of activation., the free energy of activation.

• More molecules have the required energy to More molecules have the required energy to More molecules have the required energy to More molecules have the required energy to reach the transition state.reach the transition state.reach the transition state.reach the transition state.

• Reaction is speeded up.Reaction is speeded up.Reaction is speeded up.Reaction is speeded up.• Essence of catalysis: specific binding of the Essence of catalysis: specific binding of the Essence of catalysis: specific binding of the Essence of catalysis: specific binding of the

transition statetransition statetransition statetransition state

Reaction coordinate diagram comparing Reaction coordinate diagram comparing Reaction coordinate diagram comparing Reaction coordinate diagram comparing enzyme-catalyzed and uncatalyzed reactions.enzyme-catalyzed and uncatalyzed reactions.enzyme-catalyzed and uncatalyzed reactions.enzyme-catalyzed and uncatalyzed reactions.

The terms The terms The terms The terms ΔΔΔΔGGGG‡‡‡‡uncatuncatuncatuncat and and and and ΔΔΔΔGGGG‡‡‡‡

catcatcatcat correspond to the activation energy for the correspond to the activation energy for the correspond to the activation energy for the correspond to the activation energy for the uncatalyzed reaction and the overall activation energy for the catalyzed reaction. uncatalyzed reaction and the overall activation energy for the catalyzed reaction. uncatalyzed reaction and the overall activation energy for the catalyzed reaction. uncatalyzed reaction and the overall activation energy for the catalyzed reaction.

The activation energy is lower when the enzyme catalyzes the reaction.The activation energy is lower when the enzyme catalyzes the reaction.The activation energy is lower when the enzyme catalyzes the reaction.The activation energy is lower when the enzyme catalyzes the reaction.

The Formation of an EnzymeThe Formation of an EnzymeThe Formation of an EnzymeThe Formation of an Enzyme––––Substrate Complex Substrate Complex Substrate Complex Substrate Complex Is the First Step in Enzymatic CatalysisIs the First Step in Enzymatic CatalysisIs the First Step in Enzymatic CatalysisIs the First Step in Enzymatic Catalysis

• Enzymes act by binding substratesEnzymes act by binding substratesEnzymes act by binding substratesEnzymes act by binding substrates---the non-covalent enzyme-substrate complex---the non-covalent enzyme-substrate complex---the non-covalent enzyme-substrate complex---the non-covalent enzyme-substrate complex is central is central is central is central

to the action of enzymesto the action of enzymesto the action of enzymesto the action of enzymes---allows thinking in terms of chemical interactions---allows thinking in terms of chemical interactions---allows thinking in terms of chemical interactions---allows thinking in terms of chemical interactions---allows development of kinetic equations---allows development of kinetic equations---allows development of kinetic equations---allows development of kinetic equations• Active siteActive siteActive siteActive site: a specific region that binds substrates: a specific region that binds substrates: a specific region that binds substrates: a specific region that binds substrates• Catalytic groupCatalytic groupCatalytic groupCatalytic group: the residues that directly participate : the residues that directly participate : the residues that directly participate : the residues that directly participate

in the reactionin the reactionin the reactionin the reaction• Enzyme accelerate reactions by facilitating the Enzyme accelerate reactions by facilitating the Enzyme accelerate reactions by facilitating the Enzyme accelerate reactions by facilitating the

formation of transition siteformation of transition siteformation of transition siteformation of transition site

Binding of a substrate to an enzyme at the active site Binding of a substrate to an enzyme at the active site Binding of a substrate to an enzyme at the active site Binding of a substrate to an enzyme at the active site The enzyme chymotrypsin, with bound substrate in red. Some The enzyme chymotrypsin, with bound substrate in red. Some The enzyme chymotrypsin, with bound substrate in red. Some The enzyme chymotrypsin, with bound substrate in red. Some key active-site amino acid residues appear as a red splotch on key active-site amino acid residues appear as a red splotch on key active-site amino acid residues appear as a red splotch on key active-site amino acid residues appear as a red splotch on

the enzyme surface.the enzyme surface.the enzyme surface.the enzyme surface.

Evidence for the Existence of Evidence for the Existence of Evidence for the Existence of Evidence for the Existence of an Enzyme an Enzyme an Enzyme an Enzyme –––– Substrate Complex (I) Substrate Complex (I) Substrate Complex (I) Substrate Complex (I)

An enzymeAn enzymeAn enzymeAn enzyme––––catalyzed reaction has a maximal velocity. At catalyzed reaction has a maximal velocity. At catalyzed reaction has a maximal velocity. At catalyzed reaction has a maximal velocity. At a sufficiently high substrate concentration, all the catalytic a sufficiently high substrate concentration, all the catalytic a sufficiently high substrate concentration, all the catalytic a sufficiently high substrate concentration, all the catalytic sites are filled and so the reaction rate cannot increase.sites are filled and so the reaction rate cannot increase.sites are filled and so the reaction rate cannot increase.sites are filled and so the reaction rate cannot increase.

An enzyme-catalyzed reaction reaches a maximal velocity.An enzyme-catalyzed reaction reaches a maximal velocity.An enzyme-catalyzed reaction reaches a maximal velocity.An enzyme-catalyzed reaction reaches a maximal velocity.

Structure of an enzyme-substrate complexStructure of an enzyme-substrate complexStructure of an enzyme-substrate complexStructure of an enzyme-substrate complex

X-ray crystallography provides images of enzyme-X-ray crystallography provides images of enzyme-X-ray crystallography provides images of enzyme-X-ray crystallography provides images of enzyme-substrate complexes.substrate complexes.substrate complexes.substrate complexes.

Evidence for the Existence of Evidence for the Existence of Evidence for the Existence of Evidence for the Existence of an Enzyme an Enzyme an Enzyme an Enzyme –––– Substrate Complex (II) Substrate Complex (II) Substrate Complex (II) Substrate Complex (II)

Fluorescence intensity of the pyridoxal phosphate prosthetic Fluorescence intensity of the pyridoxal phosphate prosthetic Fluorescence intensity of the pyridoxal phosphate prosthetic Fluorescence intensity of the pyridoxal phosphate prosthetic group at the active site of tryptophan synthetase:group at the active site of tryptophan synthetase:group at the active site of tryptophan synthetase:group at the active site of tryptophan synthetase:

Serine + indole Serine + indole Serine + indole Serine + indole ���� Tryptophan Tryptophan Tryptophan Tryptophan

The spectroscopic characteristics of many enzymes and substrates The spectroscopic characteristics of many enzymes and substrates The spectroscopic characteristics of many enzymes and substrates The spectroscopic characteristics of many enzymes and substrates change on formation of an enzyme-substrate complex.change on formation of an enzyme-substrate complex.change on formation of an enzyme-substrate complex.change on formation of an enzyme-substrate complex.

Evidence for the Existence of Evidence for the Existence of Evidence for the Existence of Evidence for the Existence of an Enzyme an Enzyme an Enzyme an Enzyme –––– Substrate Complex (III) Substrate Complex (III) Substrate Complex (III) Substrate Complex (III)

Active sites include distant residues (Lysozyme)Active sites include distant residues (Lysozyme)Active sites include distant residues (Lysozyme)Active sites include distant residues (Lysozyme)

1. The active site is a 3D cleft formed by groups that come 1. The active site is a 3D cleft formed by groups that come 1. The active site is a 3D cleft formed by groups that come 1. The active site is a 3D cleft formed by groups that come from different parts of the amino acid sequencefrom different parts of the amino acid sequencefrom different parts of the amino acid sequencefrom different parts of the amino acid sequence

The Active Sites of Enzymes Have Some Common FeaturesThe Active Sites of Enzymes Have Some Common FeaturesThe Active Sites of Enzymes Have Some Common FeaturesThe Active Sites of Enzymes Have Some Common Features

2. The active site takes up a relatively small part of the 2. The active site takes up a relatively small part of the 2. The active site takes up a relatively small part of the 2. The active site takes up a relatively small part of the total volume of an enzyme. The total volume of an enzyme. The total volume of an enzyme. The total volume of an enzyme. The ‘‘‘‘extraextraextraextra’’’’ amino acids amino acids amino acids amino acids serve as a scaffold to create the 3D active site from serve as a scaffold to create the 3D active site from serve as a scaffold to create the 3D active site from serve as a scaffold to create the 3D active site from amino acids that are far apart in the primary amino acids that are far apart in the primary amino acids that are far apart in the primary amino acids that are far apart in the primary structure.structure.structure.structure.

(scaffold)

3. Active sites are clefts (lined by mostly nonpolar residues). 3. Active sites are clefts (lined by mostly nonpolar residues). 3. Active sites are clefts (lined by mostly nonpolar residues). 3. Active sites are clefts (lined by mostly nonpolar residues).

Active sites

4. Substrates are bound to enzymes by multiple weak 4. Substrates are bound to enzymes by multiple weak 4. Substrates are bound to enzymes by multiple weak 4. Substrates are bound to enzymes by multiple weak attractions.attractions.attractions.attractions.

Hydrogen bonds between an enzyme and substrateHydrogen bonds between an enzyme and substrateHydrogen bonds between an enzyme and substrateHydrogen bonds between an enzyme and substrate

5. The specificity of binding depends on the precisely 5. The specificity of binding depends on the precisely 5. The specificity of binding depends on the precisely 5. The specificity of binding depends on the precisely defined arrangement of atoms in an active site.defined arrangement of atoms in an active site.defined arrangement of atoms in an active site.defined arrangement of atoms in an active site.

Substrates are bound to enzymes by multiple weak Substrates are bound to enzymes by multiple weak Substrates are bound to enzymes by multiple weak Substrates are bound to enzymes by multiple weak interactions.interactions.interactions.interactions. Because the enzyme and the substrate Because the enzyme and the substrate Because the enzyme and the substrate Because the enzyme and the substrate interact by means of short-range forces that require interact by means of short-range forces that require interact by means of short-range forces that require interact by means of short-range forces that require close contact, a substrate must have a matching shape close contact, a substrate must have a matching shape close contact, a substrate must have a matching shape close contact, a substrate must have a matching shape to fit into the site.to fit into the site.to fit into the site.to fit into the site.

� ““““Lock and KeyLock and KeyLock and KeyLock and Key”””” model (Emil Fischer, 1890) model (Emil Fischer, 1890) model (Emil Fischer, 1890) model (Emil Fischer, 1890) � ““““Induced-FitInduced-FitInduced-FitInduced-Fit”””” model (Daniel E. Koshland, Jr., 1958): model (Daniel E. Koshland, Jr., 1958): model (Daniel E. Koshland, Jr., 1958): model (Daniel E. Koshland, Jr., 1958): The active site of enzymes assume a shape that is The active site of enzymes assume a shape that is The active site of enzymes assume a shape that is The active site of enzymes assume a shape that is

complementary to that of the transition state only after the complementary to that of the transition state only after the complementary to that of the transition state only after the complementary to that of the transition state only after the substrate is bound. substrate is bound. substrate is bound. substrate is bound.

Lock-and-key model of enzyme-substrate binding Lock-and-key model of enzyme-substrate binding Lock-and-key model of enzyme-substrate binding Lock-and-key model of enzyme-substrate binding In this model, the active site of the unbound enzyme is In this model, the active site of the unbound enzyme is In this model, the active site of the unbound enzyme is In this model, the active site of the unbound enzyme is

complementary in shape to the substrate.complementary in shape to the substrate.complementary in shape to the substrate.complementary in shape to the substrate.

Induced-Fit model of enzyme-substrate binding Induced-Fit model of enzyme-substrate binding Induced-Fit model of enzyme-substrate binding Induced-Fit model of enzyme-substrate binding In this model, the enzyme changes shape on substrate In this model, the enzyme changes shape on substrate In this model, the enzyme changes shape on substrate In this model, the enzyme changes shape on substrate

binding. The active site forms a shape complementary to binding. The active site forms a shape complementary to binding. The active site forms a shape complementary to binding. The active site forms a shape complementary to the substrate only after the substrate has been bound.the substrate only after the substrate has been bound.the substrate only after the substrate has been bound.the substrate only after the substrate has been bound.

Enzymes is complementary to the Enzymes is complementary to the Enzymes is complementary to the Enzymes is complementary to the reaction transition statereaction transition statereaction transition statereaction transition state

The idea was proposed by The idea was proposed by The idea was proposed by The idea was proposed by Linus PaulingLinus PaulingLinus PaulingLinus Pauling in 1946: in 1946: in 1946: in 1946:– EnzymeEnzymeEnzymeEnzyme’’’’s active sites are complimentary to the s active sites are complimentary to the s active sites are complimentary to the s active sites are complimentary to the

transition state of the reactiontransition state of the reactiontransition state of the reactiontransition state of the reaction– Optimal interactions between substrate and Optimal interactions between substrate and Optimal interactions between substrate and Optimal interactions between substrate and

enzyme occur only in the transition state enzyme occur only in the transition state enzyme occur only in the transition state enzyme occur only in the transition state – stronger interactions with the transition state stronger interactions with the transition state stronger interactions with the transition state stronger interactions with the transition state

as compared to the ground state lower the as compared to the ground state lower the as compared to the ground state lower the as compared to the ground state lower the activation energyactivation energyactivation energyactivation energy

An imaginary enzyme (stickase) designed to An imaginary enzyme (stickase) designed to An imaginary enzyme (stickase) designed to An imaginary enzyme (stickase) designed to catalyze breakage of a metal stickcatalyze breakage of a metal stickcatalyze breakage of a metal stickcatalyze breakage of a metal stick

Weak binding interactions between the enzyme and the substrate Weak binding interactions between the enzyme and the substrate Weak binding interactions between the enzyme and the substrate Weak binding interactions between the enzyme and the substrate provide a substantial driving force for enzymatic catalysis.provide a substantial driving force for enzymatic catalysis.provide a substantial driving force for enzymatic catalysis.provide a substantial driving force for enzymatic catalysis.

Binding Energy Contributes to Binding Energy Contributes to Binding Energy Contributes to Binding Energy Contributes to Reaction Specificity and Catalysis Reaction Specificity and Catalysis Reaction Specificity and Catalysis Reaction Specificity and Catalysis

• Enzyme -catalyzed reactions are characterized by Enzyme -catalyzed reactions are characterized by Enzyme -catalyzed reactions are characterized by Enzyme -catalyzed reactions are characterized by the formation of a complex between substrate and the formation of a complex between substrate and the formation of a complex between substrate and the formation of a complex between substrate and enzyme (ES complex)enzyme (ES complex)enzyme (ES complex)enzyme (ES complex)

• Weak binding interactions between the enzyme Weak binding interactions between the enzyme Weak binding interactions between the enzyme Weak binding interactions between the enzyme and the substrate provide a substantial driving and the substrate provide a substantial driving and the substrate provide a substantial driving and the substrate provide a substantial driving force foe enzymatic catalysisforce foe enzymatic catalysisforce foe enzymatic catalysisforce foe enzymatic catalysis

• The same binding energy also gives an enzyme its The same binding energy also gives an enzyme its The same binding energy also gives an enzyme its The same binding energy also gives an enzyme its specificity, which is derived from the formation of specificity, which is derived from the formation of specificity, which is derived from the formation of specificity, which is derived from the formation of many weak binding interactions between the many weak binding interactions between the many weak binding interactions between the many weak binding interactions between the enzyme and the substrateenzyme and the substrateenzyme and the substrateenzyme and the substrate

• The need for multiple interactions is one reason The need for multiple interactions is one reason The need for multiple interactions is one reason The need for multiple interactions is one reason for the large size of enzymes for the large size of enzymes for the large size of enzymes for the large size of enzymes

Role of binding energy in catalysisRole of binding energy in catalysisRole of binding energy in catalysisRole of binding energy in catalysisTo lower the activation energy for a reaction, the system must acquire an amount To lower the activation energy for a reaction, the system must acquire an amount To lower the activation energy for a reaction, the system must acquire an amount To lower the activation energy for a reaction, the system must acquire an amount of energy equivalent to the amount by which of energy equivalent to the amount by which of energy equivalent to the amount by which of energy equivalent to the amount by which ΔΔΔΔGGGG‡‡‡‡ is lowered. is lowered. is lowered. is lowered. Much of this energy Much of this energy Much of this energy Much of this energy

comes from binding energy (comes from binding energy (comes from binding energy (comes from binding energy (ΔΔΔΔGGGGBBBB) contributed by formation of weak ) contributed by formation of weak ) contributed by formation of weak ) contributed by formation of weak noncovalent interactions between substrate and enzyme in the transition state.noncovalent interactions between substrate and enzyme in the transition state.noncovalent interactions between substrate and enzyme in the transition state.noncovalent interactions between substrate and enzyme in the transition state.

3. Enzyme Kinetics3. Enzyme Kinetics3. Enzyme Kinetics3. Enzyme Kinetics• Kinetics is the study of the rate of a reaction and Kinetics is the study of the rate of a reaction and Kinetics is the study of the rate of a reaction and Kinetics is the study of the rate of a reaction and

how it changes in response to changes in how it changes in response to changes in how it changes in response to changes in how it changes in response to changes in experimental parametersexperimental parametersexperimental parametersexperimental parameters

• Rate of enzymatic reaction is affected byRate of enzymatic reaction is affected byRate of enzymatic reaction is affected byRate of enzymatic reaction is affected by– EnzymeEnzymeEnzymeEnzyme– SubstrateSubstrateSubstrateSubstrate– EffectorsEffectorsEffectorsEffectors– TemperatureTemperatureTemperatureTemperature

Why Study Enzyme Kinetics?Why Study Enzyme Kinetics?Why Study Enzyme Kinetics?Why Study Enzyme Kinetics?

• Quantitative description of biocatalysisQuantitative description of biocatalysisQuantitative description of biocatalysisQuantitative description of biocatalysis• Determine the order of binding of substratesDetermine the order of binding of substratesDetermine the order of binding of substratesDetermine the order of binding of substrates• Understand catalytic mechanismUnderstand catalytic mechanismUnderstand catalytic mechanismUnderstand catalytic mechanism• Find effective inhibitorsFind effective inhibitorsFind effective inhibitorsFind effective inhibitors• Understand regulation of activityUnderstand regulation of activityUnderstand regulation of activityUnderstand regulation of activity

The Michaelis-Menten Model accounts for the The Michaelis-Menten Model accounts for the The Michaelis-Menten Model accounts for the The Michaelis-Menten Model accounts for the kinetic properties of many enzymeskinetic properties of many enzymeskinetic properties of many enzymeskinetic properties of many enzymes

Derivation of Enzyme Kinetics Derivation of Enzyme Kinetics Derivation of Enzyme Kinetics Derivation of Enzyme Kinetics EquationsEquationsEquationsEquations

• Start with a model mechanismStart with a model mechanismStart with a model mechanismStart with a model mechanism• Identify constraints and assumptionsIdentify constraints and assumptionsIdentify constraints and assumptionsIdentify constraints and assumptions• Carry out algebra ...Carry out algebra ...Carry out algebra ...Carry out algebra ...

– ... or graph theory for complex reactions... or graph theory for complex reactions... or graph theory for complex reactions... or graph theory for complex reactions

Simplest Model Mechanism: E + S Simplest Model Mechanism: E + S Simplest Model Mechanism: E + S Simplest Model Mechanism: E + S →→→→ ES ES ES ES →→→→ E + P E + P E + P E + P- One reactant, one product, no inhibitors- One reactant, one product, no inhibitors- One reactant, one product, no inhibitors- One reactant, one product, no inhibitors

←

Kinetic Description of Enzymatic ActivityKinetic Description of Enzymatic ActivityKinetic Description of Enzymatic ActivityKinetic Description of Enzymatic Activity

Consider an enzyme that catalyzes the S to P Consider an enzyme that catalyzes the S to P Consider an enzyme that catalyzes the S to P Consider an enzyme that catalyzes the S to P by the following pathway: by the following pathway: by the following pathway: by the following pathway:

Enzyme kinetics are more easily approached if we can ignore Enzyme kinetics are more easily approached if we can ignore Enzyme kinetics are more easily approached if we can ignore Enzyme kinetics are more easily approached if we can ignore the reverse reaction. We define the reverse reaction. We define the reverse reaction. We define the reverse reaction. We define VVVVoooo as the rate of catalysis (the as the rate of catalysis (the as the rate of catalysis (the as the rate of catalysis (the rate of increase in product with time) when [P] is low; that is, rate of increase in product with time) when [P] is low; that is, rate of increase in product with time) when [P] is low; that is, rate of increase in product with time) when [P] is low; that is, at times close to zero (Vat times close to zero (Vat times close to zero (Vat times close to zero (Voooo: initial velocity): initial velocity): initial velocity): initial velocity)

Initial velocities of enzyme-catalyzed reactionsInitial velocities of enzyme-catalyzed reactionsInitial velocities of enzyme-catalyzed reactionsInitial velocities of enzyme-catalyzed reactionsA tangent to each curve taken at time = 0 defines the A tangent to each curve taken at time = 0 defines the A tangent to each curve taken at time = 0 defines the A tangent to each curve taken at time = 0 defines the

initial velocity, initial velocity, initial velocity, initial velocity, VVVV0000, of each reaction., of each reaction., of each reaction., of each reaction.

Changes in the concentration of reaction participants of Changes in the concentration of reaction participants of Changes in the concentration of reaction participants of Changes in the concentration of reaction participants of an enzyme-catalyzed reaction with timean enzyme-catalyzed reaction with timean enzyme-catalyzed reaction with timean enzyme-catalyzed reaction with time

Concentration changes under (A) steady-state conditions, Concentration changes under (A) steady-state conditions, Concentration changes under (A) steady-state conditions, Concentration changes under (A) steady-state conditions, and (B) the pre-steady-state conditions.and (B) the pre-steady-state conditions.and (B) the pre-steady-state conditions.and (B) the pre-steady-state conditions.

We want to express [ES] in terms of known quantities We want to express [ES] in terms of known quantities We want to express [ES] in terms of known quantities We want to express [ES] in terms of known quantities

How to get the Michaelis-Menten EquationHow to get the Michaelis-Menten EquationHow to get the Michaelis-Menten EquationHow to get the Michaelis-Menten Equation

At steady-state:At steady-state:At steady-state:At steady-state:rate of formation (ES) = rate of breakdown (ES)rate of formation (ES) = rate of breakdown (ES)rate of formation (ES) = rate of breakdown (ES)rate of formation (ES) = rate of breakdown (ES)

defined as Michaelis constant (defined as Michaelis constant (defined as Michaelis constant (defined as Michaelis constant (KKKKMMMM), ), ), ), has the unit of concentrationhas the unit of concentrationhas the unit of concentrationhas the unit of concentration

The maximal rate, VThe maximal rate, VThe maximal rate, VThe maximal rate, Vmaxmaxmaxmax, is attained when the catalytic , is attained when the catalytic , is attained when the catalytic , is attained when the catalytic sites on the enzyme are saturated with substrate --- that sites on the enzyme are saturated with substrate --- that sites on the enzyme are saturated with substrate --- that sites on the enzyme are saturated with substrate --- that is, when [ES] = [E]is, when [ES] = [E]is, when [ES] = [E]is, when [ES] = [E]TTTT. . . .

Effect of substrate concentration on the Effect of substrate concentration on the Effect of substrate concentration on the Effect of substrate concentration on the initial velocity of an enzyme-catalyzed reactioninitial velocity of an enzyme-catalyzed reactioninitial velocity of an enzyme-catalyzed reactioninitial velocity of an enzyme-catalyzed reaction

The Michaelis constant (The Michaelis constant (The Michaelis constant (The Michaelis constant (KKKKMMMM) is the substrate concentration ) is the substrate concentration ) is the substrate concentration ) is the substrate concentration yielding a velocity of Vyielding a velocity of Vyielding a velocity of Vyielding a velocity of Vmaxmaxmaxmax/2./2./2./2.

Dependence of initial velocity on substrate concentrationDependence of initial velocity on substrate concentrationDependence of initial velocity on substrate concentrationDependence of initial velocity on substrate concentration

At low [S], [S] << [KAt low [S], [S] << [KAt low [S], [S] << [KAt low [S], [S] << [KMMMM]: V]: V]: V]: Voooo= V= V= V= Vmaxmaxmaxmax [S]/K [S]/K [S]/K [S]/KM M M M ; that is, V; that is, V; that is, V; that is, Voooo ∝[S][S][S][S]

Dissection of the Michaelis Dissection of the Michaelis Dissection of the Michaelis Dissection of the Michaelis –––– Menten Equation Menten Equation Menten Equation Menten Equation

At high [S], [S] >> [KAt high [S], [S] >> [KAt high [S], [S] >> [KAt high [S], [S] >> [KMMMM]: V]: V]: V]: Voooo= V= V= V= Vmaxmaxmaxmax

Determine Determine Determine Determine KKKKMMMM and V and V and V and Vmax max max max by Double-reciprocal Plotsby Double-reciprocal Plotsby Double-reciprocal Plotsby Double-reciprocal Plots

Double-reciprocal plot of Double-reciprocal plot of Double-reciprocal plot of Double-reciprocal plot of enzyme kinetics is generated enzyme kinetics is generated enzyme kinetics is generated enzyme kinetics is generated by plotting 1/Vby plotting 1/Vby plotting 1/Vby plotting 1/V0000 as a function as a function as a function as a function 1/[S]. 1/[S]. 1/[S]. 1/[S]. The slope is the The slope is the The slope is the The slope is the KKKKMMMM/V/V/V/Vmaxmaxmaxmax, the intercept on the , the intercept on the , the intercept on the , the intercept on the vertical axis is 1/Vvertical axis is 1/Vvertical axis is 1/Vvertical axis is 1/Vmaxmaxmaxmax, and , and , and , and the intercept on the the intercept on the the intercept on the the intercept on the horizontal axis is -1/horizontal axis is -1/horizontal axis is -1/horizontal axis is -1/KKKKMMMM....

Physical Consequence of Physical Consequence of Physical Consequence of Physical Consequence of KKKKMMMM(Sensitivity of Individuals to Ethanol)(Sensitivity of Individuals to Ethanol)(Sensitivity of Individuals to Ethanol)(Sensitivity of Individuals to Ethanol)

Excess CH3CHO Facial flushing,Facial flushing,Facial flushing,Facial flushing,rapid heart raterapid heart raterapid heart raterapid heart rate

…………

Normally, the acetaldehyde, which is the cause of the Normally, the acetaldehyde, which is the cause of the Normally, the acetaldehyde, which is the cause of the Normally, the acetaldehyde, which is the cause of the symptoms when present at high concentrations, is processed symptoms when present at high concentrations, is processed symptoms when present at high concentrations, is processed symptoms when present at high concentrations, is processed to acetate by acetaldehyde dehydrogenase.to acetate by acetaldehyde dehydrogenase.to acetate by acetaldehyde dehydrogenase.to acetate by acetaldehyde dehydrogenase.

Two forms of acetaldehyde dehydrogenase:Two forms of acetaldehyde dehydrogenase:Two forms of acetaldehyde dehydrogenase:Two forms of acetaldehyde dehydrogenase:1. Cytosolic: high 1. Cytosolic: high 1. Cytosolic: high 1. Cytosolic: high KKKKM M M M ���� high concentration of substrate is high concentration of substrate is high concentration of substrate is high concentration of substrate is

needed for the enzyme to achieve (needed for the enzyme to achieve (needed for the enzyme to achieve (needed for the enzyme to achieve (VVVVmaxmaxmaxmax /2) /2) /2) /2)2. Mitochondrial: low 2. Mitochondrial: low 2. Mitochondrial: low 2. Mitochondrial: low KKKKM M M M ���� low concentration of substrate is low concentration of substrate is low concentration of substrate is low concentration of substrate is

needed for the enzyme to achieve (needed for the enzyme to achieve (needed for the enzyme to achieve (needed for the enzyme to achieve (VVVVmaxmaxmaxmax /2) /2) /2) /2)

For a certain amount of CHFor a certain amount of CHFor a certain amount of CHFor a certain amount of CH3333CHO, conversion into CHO, conversion into CHO, conversion into CHO, conversion into CHCHCHCH3333COOCOOCOOCOO---- by the mitochondrial form is faster. (In certain by the mitochondrial form is faster. (In certain by the mitochondrial form is faster. (In certain by the mitochondrial form is faster. (In certain time, less CHtime, less CHtime, less CHtime, less CH3333CHO is converted by the cytosolic form.)CHO is converted by the cytosolic form.)CHO is converted by the cytosolic form.)CHO is converted by the cytosolic form.)

In some individuals, the mitochondrial form is mutated and In some individuals, the mitochondrial form is mutated and In some individuals, the mitochondrial form is mutated and In some individuals, the mitochondrial form is mutated and thus is less active. Since the cytosolic form has higher thus is less active. Since the cytosolic form has higher thus is less active. Since the cytosolic form has higher thus is less active. Since the cytosolic form has higher KKKKMMMM, , , , which means that more CHwhich means that more CHwhich means that more CHwhich means that more CH3333CHO is accumulated, therefore, CHO is accumulated, therefore, CHO is accumulated, therefore, CHO is accumulated, therefore, these people are more susceptible to facial flushing and these people are more susceptible to facial flushing and these people are more susceptible to facial flushing and these people are more susceptible to facial flushing and rapid heart rate after alcohol intake.rapid heart rate after alcohol intake.rapid heart rate after alcohol intake.rapid heart rate after alcohol intake.

Significance of Significance of Significance of Significance of KKKKMMMM Value Value Value Value� KKKKMMMM provides a measure of the substrate concentration provides a measure of the substrate concentration provides a measure of the substrate concentration provides a measure of the substrate concentration

required for significant catalysis to occurrequired for significant catalysis to occurrequired for significant catalysis to occurrequired for significant catalysis to occur

� KKKKMMMM can be used as a measure of the enzyme can be used as a measure of the enzyme can be used as a measure of the enzyme can be used as a measure of the enzyme’’’’s binding s binding s binding s binding affinity when kaffinity when kaffinity when kaffinity when k2222 <<k <<k <<k <<k-1 -1 -1 -1 （ KKKKMMMM=k=k=k=k-1-1-1-1/k/k/k/k1111）

� For many enzymes, experimental evidence suggests For many enzymes, experimental evidence suggests For many enzymes, experimental evidence suggests For many enzymes, experimental evidence suggests that that that that KKKKMMMM provides an approximation of substrate provides an approximation of substrate provides an approximation of substrate provides an approximation of substrate concentration concentration concentration concentration in vivoin vivoin vivoin vivo

� When When When When KKKKMMMM is known, the fraction of sites filled, f is known, the fraction of sites filled, f is known, the fraction of sites filled, f is known, the fraction of sites filled, fESESESES, at , at , at , at any substrate concentration can be calculated fromany substrate concentration can be calculated fromany substrate concentration can be calculated fromany substrate concentration can be calculated from

The The The The KKKKM M M M value for an enzyme depends on the value for an enzyme depends on the value for an enzyme depends on the value for an enzyme depends on the particular substrate and on environmental particular substrate and on environmental particular substrate and on environmental particular substrate and on environmental conditions such as pH, temperature, and ionic strength.conditions such as pH, temperature, and ionic strength.conditions such as pH, temperature, and ionic strength.conditions such as pH, temperature, and ionic strength.

Significance of VSignificance of VSignificance of VSignificance of Vmaxmaxmaxmax Value Value Value Value VVVVmaxmaxmaxmax reveals the turnover number of an enzyme ( reveals the turnover number of an enzyme ( reveals the turnover number of an enzyme ( reveals the turnover number of an enzyme (kkkk2222, also , also , also , also called called called called kkkkcatcatcatcat), which is the number of substrate molecules ), which is the number of substrate molecules ), which is the number of substrate molecules ), which is the number of substrate molecules converted into product by an enzyme molecule in a unit converted into product by an enzyme molecule in a unit converted into product by an enzyme molecule in a unit converted into product by an enzyme molecule in a unit time when the enzyme is fully saturated with substrate. time when the enzyme is fully saturated with substrate. time when the enzyme is fully saturated with substrate. time when the enzyme is fully saturated with substrate.

VVVVmax max max max is achieved when [ES]=[E]is achieved when [ES]=[E]is achieved when [ES]=[E]is achieved when [ES]=[E]TTTT. Thus. Thus. Thus. Thus

Under physiological conditions, the [S]/KUnder physiological conditions, the [S]/KUnder physiological conditions, the [S]/KUnder physiological conditions, the [S]/KMMMM ratio is ratio is ratio is ratio is typically between 0.01 and 1.0, Vtypically between 0.01 and 1.0, Vtypically between 0.01 and 1.0, Vtypically between 0.01 and 1.0, Vmaxmaxmaxmax is not achieved. is not achieved. is not achieved. is not achieved.

When [S] << KWhen [S] << KWhen [S] << KWhen [S] << KMMMM, [E]=[E], [E]=[E], [E]=[E], [E]=[E]TTTT. Thus. Thus. Thus. Thus

Kinetic Perfection in Enzymatic Catalysis:Kinetic Perfection in Enzymatic Catalysis:Kinetic Perfection in Enzymatic Catalysis:Kinetic Perfection in Enzymatic Catalysis:The The The The kkkkcatcatcatcat////KKKKMMMM Criterion Criterion Criterion Criterion

� When [S] << When [S] << When [S] << When [S] << KKKKMMMM, , , , kkkkcatcatcatcat////KKKKMMMM is the rate constant is the rate constant is the rate constant is the rate constant for the interaction of E and Sfor the interaction of E and Sfor the interaction of E and Sfor the interaction of E and S

� kkkkcatcatcatcat////KKKKM M M M can be used as a measure of catalytic can be used as a measure of catalytic can be used as a measure of catalytic can be used as a measure of catalytic efficiencyefficiencyefficiencyefficiency

S + ES + ES + ES + E E + PE + PE + PE + Pkkkkcatcatcatcat////KKKKMMMM

Chymotrypsin has a preference for cleaving next to Chymotrypsin has a preference for cleaving next to Chymotrypsin has a preference for cleaving next to Chymotrypsin has a preference for cleaving next to bulky, hydrophobic side chains.bulky, hydrophobic side chains.bulky, hydrophobic side chains.bulky, hydrophobic side chains.

How efficient can an enzyme be? How efficient can an enzyme be? How efficient can an enzyme be? How efficient can an enzyme be? (How high can (How high can (How high can (How high can kkkkcatcatcatcat////KKKKM M M M be?) be?) be?) be?)

Substituting for Substituting for Substituting for Substituting for KKKKMMMM gives gives gives gives

The ultimate limit on the value of The ultimate limit on the value of The ultimate limit on the value of The ultimate limit on the value of kkkkcatcatcatcat////KKKKMMMM is set by is set by is set by is set by kkkk1111, the rate , the rate , the rate , the rate constant of formation of the ES complex. This rate constant constant of formation of the ES complex. This rate constant constant of formation of the ES complex. This rate constant constant of formation of the ES complex. This rate constant cannot be faster than the diffusion-controlled encounter of an cannot be faster than the diffusion-controlled encounter of an cannot be faster than the diffusion-controlled encounter of an cannot be faster than the diffusion-controlled encounter of an enzyme and its substrate.enzyme and its substrate.enzyme and its substrate.enzyme and its substrate.The ratio The ratio The ratio The ratio kkkkcatcatcatcat////KKKKMMMM provides a good measure of catalytic provides a good measure of catalytic provides a good measure of catalytic provides a good measure of catalytic efficiency efficiency efficiency efficiency

Kinetic PerfectionKinetic PerfectionKinetic PerfectionKinetic PerfectionEnzymes such as these that have Enzymes such as these that have Enzymes such as these that have Enzymes such as these that have kkkkcatcatcatcat////KKKKMMMM ratios at ratios at ratios at ratios at the upper limits have attained kinetic perfection. the upper limits have attained kinetic perfection. the upper limits have attained kinetic perfection. the upper limits have attained kinetic perfection. Their catalytic velocity is restricted only by the rate Their catalytic velocity is restricted only by the rate Their catalytic velocity is restricted only by the rate Their catalytic velocity is restricted only by the rate at which they encounter substrate in the solution.at which they encounter substrate in the solution.at which they encounter substrate in the solution.at which they encounter substrate in the solution.

How to Do Kinetic MeasurementsHow to Do Kinetic MeasurementsHow to Do Kinetic MeasurementsHow to Do Kinetic Measurements

Determination of Kinetic ParametersDetermination of Kinetic ParametersDetermination of Kinetic ParametersDetermination of Kinetic Parameters

Nonlinear Michaelis-Menten plot should be used to Nonlinear Michaelis-Menten plot should be used to Nonlinear Michaelis-Menten plot should be used to Nonlinear Michaelis-Menten plot should be used to calculate parameters calculate parameters calculate parameters calculate parameters KKKKmmmm and and and and VVVVmaxmaxmaxmax

Linearized double-reciprocal plot is good for Linearized double-reciprocal plot is good for Linearized double-reciprocal plot is good for Linearized double-reciprocal plot is good for analysis of two-substrate data or inhibition analysis of two-substrate data or inhibition analysis of two-substrate data or inhibition analysis of two-substrate data or inhibition

Effect of Substrate ConcentrationEffect of Substrate ConcentrationEffect of Substrate ConcentrationEffect of Substrate Concentration

• Ideal Rate: Ideal Rate: Ideal Rate: Ideal Rate:

• Deviations due to: Deviations due to: Deviations due to: Deviations due to:

–Limitation of measurementsLimitation of measurementsLimitation of measurementsLimitation of measurements

–Substrate inhibitionSubstrate inhibitionSubstrate inhibitionSubstrate inhibition

–Substrate prep contains inhibitorsSubstrate prep contains inhibitorsSubstrate prep contains inhibitorsSubstrate prep contains inhibitors

–Enzyme prep contains inhibitorsEnzyme prep contains inhibitorsEnzyme prep contains inhibitorsEnzyme prep contains inhibitors

SKSVv

m +=

][max

Two-substrate ReactionsTwo-substrate ReactionsTwo-substrate ReactionsTwo-substrate Reactions• Kinetic mechanismKinetic mechanismKinetic mechanismKinetic mechanism: the order of binding of substrates : the order of binding of substrates : the order of binding of substrates : the order of binding of substrates

and release of productsand release of productsand release of productsand release of products

• When two or more reactants are involved, enzyme When two or more reactants are involved, enzyme When two or more reactants are involved, enzyme When two or more reactants are involved, enzyme kinetics allows to distinguish between different kinetic kinetics allows to distinguish between different kinetic kinetics allows to distinguish between different kinetic kinetics allows to distinguish between different kinetic mechanismsmechanismsmechanismsmechanisms

• Sequential mechanism (ternary complex)Sequential mechanism (ternary complex)Sequential mechanism (ternary complex)Sequential mechanism (ternary complex)

• Ping-Pong mechanism (double-displacement)Ping-Pong mechanism (double-displacement)Ping-Pong mechanism (double-displacement)Ping-Pong mechanism (double-displacement)

Common mechanisms for enzyme-catalyzed bisubstrate reactionsCommon mechanisms for enzyme-catalyzed bisubstrate reactionsCommon mechanisms for enzyme-catalyzed bisubstrate reactionsCommon mechanisms for enzyme-catalyzed bisubstrate reactions

Steady-state kinetic analysis of bisubstrate reactions Steady-state kinetic analysis of bisubstrate reactions Steady-state kinetic analysis of bisubstrate reactions Steady-state kinetic analysis of bisubstrate reactionsIntersecting lines indicate that a ternary complex is Intersecting lines indicate that a ternary complex is Intersecting lines indicate that a ternary complex is Intersecting lines indicate that a ternary complex is

formed in the reactionformed in the reactionformed in the reactionformed in the reaction

Steady-state kinetic analysis of bisubstrate reactionsSteady-state kinetic analysis of bisubstrate reactionsSteady-state kinetic analysis of bisubstrate reactionsSteady-state kinetic analysis of bisubstrate reactionsParallel lines indicate a Ping-Pong Parallel lines indicate a Ping-Pong Parallel lines indicate a Ping-Pong Parallel lines indicate a Ping-Pong

(double-displacement) pathway(double-displacement) pathway(double-displacement) pathway(double-displacement) pathway

Enzyme Inhibitors Are Compounds Enzyme Inhibitors Are Compounds Enzyme Inhibitors Are Compounds Enzyme Inhibitors Are Compounds That Decrease EnzymeThat Decrease EnzymeThat Decrease EnzymeThat Decrease Enzyme’’’’s Activitys Activitys Activitys Activity

• Irreversible inhibitors Irreversible inhibitors Irreversible inhibitors Irreversible inhibitors (inactivators) react with the enzyme(inactivators) react with the enzyme(inactivators) react with the enzyme(inactivators) react with the enzyme- one inhibitor molecule can permanently shut off one enzyme one inhibitor molecule can permanently shut off one enzyme one inhibitor molecule can permanently shut off one enzyme one inhibitor molecule can permanently shut off one enzyme moleculemoleculemoleculemolecule- they are often they are often they are often they are often powerful toxinspowerful toxinspowerful toxinspowerful toxins but also may be used as drugs but also may be used as drugs but also may be used as drugs but also may be used as drugs

• Reversible inhibitors Reversible inhibitors Reversible inhibitors Reversible inhibitors bind to, and can dissociate from the bind to, and can dissociate from the bind to, and can dissociate from the bind to, and can dissociate from the enzymeenzymeenzymeenzyme

- they are often structural analogs of substrates or products - they are often structural analogs of substrates or products - they are often structural analogs of substrates or products - they are often structural analogs of substrates or products - they are often - they are often - they are often - they are often used as drugsused as drugsused as drugsused as drugs to slow down a specific enzyme to slow down a specific enzyme to slow down a specific enzyme to slow down a specific enzyme - may be competitive, uncompetitive, or mixed - may be competitive, uncompetitive, or mixed - may be competitive, uncompetitive, or mixed - may be competitive, uncompetitive, or mixed

Classification of Reversible Classification of Reversible Classification of Reversible Classification of Reversible InhibitorsInhibitorsInhibitorsInhibitors

Reversible inhibitor can bind: Reversible inhibitor can bind: Reversible inhibitor can bind: Reversible inhibitor can bind: – To the free enzyme and prevent the To the free enzyme and prevent the To the free enzyme and prevent the To the free enzyme and prevent the

binding of the substratebinding of the substratebinding of the substratebinding of the substrate– To the enzyme-substrate complex and To the enzyme-substrate complex and To the enzyme-substrate complex and To the enzyme-substrate complex and

prevent the reaction prevent the reaction prevent the reaction prevent the reaction

Competitive inhibitors bind to the enzyme's active site; Competitive inhibitors bind to the enzyme's active site; Competitive inhibitors bind to the enzyme's active site; Competitive inhibitors bind to the enzyme's active site; KKKKIIII is the equilibrium constant for inhibitor binding to E. is the equilibrium constant for inhibitor binding to E. is the equilibrium constant for inhibitor binding to E. is the equilibrium constant for inhibitor binding to E.

Lines intersect at the y-axisLines intersect at the y-axisLines intersect at the y-axisLines intersect at the y-axis

Uncompetitive inhibitors bind at a separate site, Uncompetitive inhibitors bind at a separate site, Uncompetitive inhibitors bind at a separate site, Uncompetitive inhibitors bind at a separate site, but bind only to the ES complex; but bind only to the ES complex; but bind only to the ES complex; but bind only to the ES complex; KKKKIIII′′′′ is the equilibrium is the equilibrium is the equilibrium is the equilibrium constant for inhibitor binding to ES.constant for inhibitor binding to ES.constant for inhibitor binding to ES.constant for inhibitor binding to ES.Lines are parallelLines are parallelLines are parallelLines are parallel

Mixed inhibitors bind at a separate site, Mixed inhibitors bind at a separate site, Mixed inhibitors bind at a separate site, Mixed inhibitors bind at a separate site, but may bind to either E or ES.but may bind to either E or ES.but may bind to either E or ES.but may bind to either E or ES.Lines intersect left from the y-axisLines intersect left from the y-axisLines intersect left from the y-axisLines intersect left from the y-axis

********************

Irreversible inhibitionIrreversible inhibitionIrreversible inhibitionIrreversible inhibitionReaction of chymotrypsin with diisopropylfluorophosphate (DIFP), which modifies SerReaction of chymotrypsin with diisopropylfluorophosphate (DIFP), which modifies SerReaction of chymotrypsin with diisopropylfluorophosphate (DIFP), which modifies SerReaction of chymotrypsin with diisopropylfluorophosphate (DIFP), which modifies Ser195195195195, , , ,

irreversibly inhibits the enzyme. This has led to the conclusion that Serirreversibly inhibits the enzyme. This has led to the conclusion that Serirreversibly inhibits the enzyme. This has led to the conclusion that Serirreversibly inhibits the enzyme. This has led to the conclusion that Ser195195195195 is the key is the key is the key is the key active-site Ser residue in chymotrypsin. active-site Ser residue in chymotrypsin. active-site Ser residue in chymotrypsin. active-site Ser residue in chymotrypsin.

Irreversible inhibitors bind Irreversible inhibitors bind Irreversible inhibitors bind Irreversible inhibitors bind covalently with or destroy a covalently with or destroy a covalently with or destroy a covalently with or destroy a functional group on an enzyme functional group on an enzyme functional group on an enzyme functional group on an enzyme that is essential for the that is essential for the that is essential for the that is essential for the enzymeenzymeenzymeenzyme’’’’s activity, or form a s activity, or form a s activity, or form a s activity, or form a particularly stable particularly stable particularly stable particularly stable noncovalent association.noncovalent association.noncovalent association.noncovalent association.

The pH-activity profiles of two enzymesThe pH-activity profiles of two enzymesThe pH-activity profiles of two enzymesThe pH-activity profiles of two enzymesThe pH optimum for the activity of an enzyme is generally close to The pH optimum for the activity of an enzyme is generally close to The pH optimum for the activity of an enzyme is generally close to The pH optimum for the activity of an enzyme is generally close to the pH of the environment in which the enzyme is normally found. the pH of the environment in which the enzyme is normally found. the pH of the environment in which the enzyme is normally found. the pH of the environment in which the enzyme is normally found.

Question IQuestion IQuestion IQuestion IThe following experimental data were collected during a study The following experimental data were collected during a study The following experimental data were collected during a study The following experimental data were collected during a study of the catalytic activity of an intestinal peptidase with the of the catalytic activity of an intestinal peptidase with the of the catalytic activity of an intestinal peptidase with the of the catalytic activity of an intestinal peptidase with the substrate Glycylglycine.substrate Glycylglycine.substrate Glycylglycine.substrate Glycylglycine.Use graphical analysis (see Box 6Use graphical analysis (see Box 6Use graphical analysis (see Box 6Use graphical analysis (see Box 6––––1 and its associated Living1 and its associated Living1 and its associated Living1 and its associated LivingGraph) to determine the Graph) to determine the Graph) to determine the Graph) to determine the KKKKmmmm and V and V and V and Vmax max max max for this enzyme for this enzyme for this enzyme for this enzyme preparation and substrate.preparation and substrate.preparation and substrate.preparation and substrate.

Question IIQuestion IIQuestion IIQuestion IICarbonic anhydrase is strongly inhibited by the drug acetazolamide, which is Carbonic anhydrase is strongly inhibited by the drug acetazolamide, which is Carbonic anhydrase is strongly inhibited by the drug acetazolamide, which is Carbonic anhydrase is strongly inhibited by the drug acetazolamide, which is used as a diuretic (i.e., to increase the production of urine) and to lower used as a diuretic (i.e., to increase the production of urine) and to lower used as a diuretic (i.e., to increase the production of urine) and to lower used as a diuretic (i.e., to increase the production of urine) and to lower excessively high pressure in the eye (due to accumulation of intraocular fluid) excessively high pressure in the eye (due to accumulation of intraocular fluid) excessively high pressure in the eye (due to accumulation of intraocular fluid) excessively high pressure in the eye (due to accumulation of intraocular fluid) in glaucoma. The experimental curve of initial reaction velocity (as in glaucoma. The experimental curve of initial reaction velocity (as in glaucoma. The experimental curve of initial reaction velocity (as in glaucoma. The experimental curve of initial reaction velocity (as percentage of percentage of percentage of percentage of Vmax) versus [S] for the carbonic Vmax) versus [S] for the carbonic Vmax) versus [S] for the carbonic Vmax) versus [S] for the carbonic anhydrase reaction is anhydrase reaction is anhydrase reaction is anhydrase reaction is illustrated below (upper curve). When the experiment is repeated in the illustrated below (upper curve). When the experiment is repeated in the illustrated below (upper curve). When the experiment is repeated in the illustrated below (upper curve). When the experiment is repeated in the presence of acetazolamide, the lower curve is obtained. Determine the nature presence of acetazolamide, the lower curve is obtained. Determine the nature presence of acetazolamide, the lower curve is obtained. Determine the nature presence of acetazolamide, the lower curve is obtained. Determine the nature of the inhibition by acetazolamide. Explain your reasoning.of the inhibition by acetazolamide. Explain your reasoning.of the inhibition by acetazolamide. Explain your reasoning.of the inhibition by acetazolamide. Explain your reasoning.

• Enzymes Are Powerful and Highly Specific Enzymes Are Powerful and Highly Specific Enzymes Are Powerful and Highly Specific Enzymes Are Powerful and Highly Specific CatalystsCatalystsCatalystsCatalysts

• Free Energy Is a Useful Thermodynamic Free Energy Is a Useful Thermodynamic Free Energy Is a Useful Thermodynamic Free Energy Is a Useful Thermodynamic Function for Understanding EnzymesFunction for Understanding EnzymesFunction for Understanding EnzymesFunction for Understanding Enzymes

• Enzymes Accelerate Reactions by Facilitating the Enzymes Accelerate Reactions by Facilitating the Enzymes Accelerate Reactions by Facilitating the Enzymes Accelerate Reactions by Facilitating the Formation of the Transition StateFormation of the Transition StateFormation of the Transition StateFormation of the Transition State

• The Michaelis-Menten Model Accounts for the The Michaelis-Menten Model Accounts for the The Michaelis-Menten Model Accounts for the The Michaelis-Menten Model Accounts for the Kinetic Properties of Many EnzymesKinetic Properties of Many EnzymesKinetic Properties of Many EnzymesKinetic Properties of Many Enzymes

• Enzymes Can Be Inhibited by Specific MoleculesEnzymes Can Be Inhibited by Specific MoleculesEnzymes Can Be Inhibited by Specific MoleculesEnzymes Can Be Inhibited by Specific Molecules

SummarySummarySummarySummary