enzymes: basic concepts and kinetics protein catalysts

Enzymes: Basic Concepts and Kinetics

Protein catalysts

I. Nomenclature

Each enzyme is assigned two names: 1. Recommended name: short, convenient; common suffix “ase” attached to the substrate (reactants) or to the action performed (e.g. proteinase, DNase; lactate dehydrogenase, adenylyl cyclase); some trivial names, e.g. pepsin 2. Systematic name: developed by IUBMB , 6 major classes, each with numerous subgroups, unambiguous and informative e.g. D-glyceraldehyde 3-phosphate: NAD oxidoreductase

II. Properties of enzymes

Protein catalysts that increase the velocity of a chemical reaction.

1. Active sites: special pocket or cleft containing aa side

chains that form a 3-D surface complementary to the substrate.

Common features of active sites

• A 3-D cleft or crevices formed by groups from different parts of amino acid sequence (Fig 3.1).

• Small part of the total volume of an enzyme.

more amino acids serve as a scaffold, regulatory sites, sites for interaction with other proteins, or the channels to bring the substrate to active sites.

• Substrates are bound to enzymes by multiple weak attractions. Noncovalent interations: electrostatic interactions, hydrogen bonds, van der Waals forces, hydrophobic interactions.

Asp101

Active siteCatalytic groups

Glu35Asp52

Asp101

Trp108

Figure 3.1 Active sites may include distant residues. (A) Ribbon diagram of the enzyme lysozyme with several components of the active site shown in color. (B) A schematic representation of the primary structure of lysozyme shows that the active site is composed of residues that come from different parts of the polypeptide chain.

Figure 3.2 Substrates are bound to enzymes by multiple weak attractions. (left) The enzyme cytochrome p-450 is illustrated bound to its substrate camphor. (right) In the active site, the substrate is surrounded by residues from the enzyme. Note also the presence of a heme cofactor.

2. Catalytic efficiency

• High efficient: 103 – 108 times faster, each enzyme molecule can transform 100-1000 substrate molecules to product per second.

• Turnover number: the number of substrate molecules converted into product by an enzyme molecule in a unit time when the enzyme is fully saturated with substrate.

3. Specificity

• High specificity: no side reactions

absolute specificity (only one substrate), relative specificity (one type of chemical bond), steric specificity (only one optical isomer)

Proteolytic enzymes (e.g. proteinases)

Figure 3.3 Enzyme specificity. (A) Trypsin cleaves on the carboxyl side of arginine and lysine residues, whereas (B) thrombin cleaves Arg-Gly bonds in particular sequences specifically.

4. Cofactors Apoenzyme + cofactor = holoenzyme

No protein cofactors:• Metal ions: Zn2+, or Fe 2+

• Organic molecules: coenzymes, derivatives of vitamins

• Holoenzyme: enzyme with its cofactor• Apoenzyme: the protein portion of the

holoenzyme• Prosthetic group: a tightly bound coenzyme

5. Regulation

Enzyme activity can be regulated in the cell:

activated or inhibited

6. Location within the cell

• In specific organelles of the cell (compartmentalization)

• Isolating the reaction substrate or product from other competing reactions

• Providing a favorable environment for the reaction

Fig 3.4 Intracellular location of some important biochemical pathways involved enzyme-catalyzed reactions.

III. The mechanism of enzymes action

1. Energy changes occurring during the reaction

energy barrier (free energy of activation) chemical reaction:

Reactant A to product B through the transition state S‡ (high energy intermediate)

A ⇌ S‡ B⇌

• Free energy of activation:

The peak of energy in Figure 3.5 is the difference in free energy (∆G‡) between the reactant and S‡ ;

Because of the high free energy of activation, the rates of uncatalyzed chemical reactions are often slow.

Figure 3.5 Enzymes decrease the activation energy.

• Rate of reaction: Molecules must overcome the energy

barrier of the transition state. The rate is determined by the energized

molecules. The lower the ∆G‡ , the faster the rate.

• Alternate reaction pathway an enzyme provides alternate reaction

pathway with a lower ∆G‡ , does not change the ∆G, and equilibrium of the reaction.

Figure 3.6 Enzymes decrease the activation energy.

2. Chemistry of the active site

• Active site: binding substrates and form a complex molecular machine employing a diversity of chemical mechanisms to facilitate the conversion of substrate to product.

Many factors related to catalytic efficiency of enzymes.

• Transition-state stabilization:

active site as a flexible molecular template for the activated transition state

Enzymes stabilize the substrate in its transition state, and increase the concentration of the reactive intermediate, thus accelerates the reaction.

• Other mechanisms: catalytic groups in active site enhance the formation of the transition state.

general acid-base catalysis

covalent enzyme-substrate complex

(E + S ES ⇌ → E + P)

Figure 3.7 Lock-and-key model of enzyme-substrate binding. In this model, the active site of the unbound enzyme is complementary in shape to the substrate.

Figure 3.8 Induced-fit model of enzyme-substrate binding. In this model, the enzyme changes shape on substrate binding. The active site forms a shape complementary to the substrate only after the substrate has been bound.

Ⅳ. Factors affecting reaction velocity

1. Substrate concentration The velocity or rate : the number of substrate molecules

converted to product per unit time.

The velocity will increase with substrate concentration until a maximal velocity (Vm) is reached (reflecting the saturation of binding sites ).

• Michaelis-Menten kinetics E + S ES ⇌ → E + P

V0= Vmax[S]/(Km+[S])

The plot of initial reaction velocity ( V0 ) against substrate concentration ([S]) is hyperbolic. Some allosteric enzymes show a sigmoidal curve.

Figure 3.9 Reaction velocity versus substrate concentration in an enzyme-catalyzed reaction. An enzyme-catalyzed reaction reaches a maximal velocity.

Figure 3.10 Kinetics for an allosteric enzyme. Allosteric enzymes display a sigmoidal dependence of reaction velocity on substrate concentration.

2. Temperature

• Increase of velocity with temperature:

increased number of activated molecules

• Decrease of velocity with higher temperature:

denaturation of protein enzymes

• Optimal temperature

most proteins in human 37ºC

Figure 3.10 Effect of temperature on an enzyme-catalyzed reaction

V

Temperature, ºC

Heat inactivation of enzyme

V

3. pH

• Effect of the pH on the ionization of the active site: the pH affects reactive groups of both enzyme and substrate in ionized or unionized state.

• Effect of pH on enzyme denaturation

extremes of pH cause protein denaturation• The pH optimum varies for different enzymes: the

pH at which maximal enzyme activity is achieved.

pepsin – pH 2, many other enzymes- pH 7.4

Figure 3.11 Effect of pH on enzyme-catalyzed reactions

V. Michaelis-Menten equation

1. Reaction model: for one substrate molecule

E + S ⇌ ES → E + P

S – the substrate, E – the enzyme ES – the enzyme-substrate complex P – the product

k1, k-1and k2 – the rate constants

k1

k-1

k2

2. Michaelis-Menten equation:

Reaction velocity varies with substrate concentration:

V0= Vmax[S] / (Km+[S])

V0 = initial reaction velocity

Vmax= maximal velocity

[S] = substrate concentration

Km = Michaelis constant = (k-1+ k2)/k1

The assumptions made in deriving the equation

• Relative concentrations of E and S: [S] is much greater than [E]. The bound substrate is a very small part of total substrate at any one time.

• Steady-state assumption: [ES] does not change with time, the rate of formation of ES is equal to that of the breakdown of ES.

• Initial velocity (V0): the rate measured as soon as enzyme and substrate are mixed. At that time, [P] is very small, the rate of back reaction from P to S

can be ignored.

3. Important conclusions about Michaelis-Menten kinetics

• Characteristics of Km:

a characteristic constant of an enzyme

affinity of the enzyme for that substrate

When Vo=1/2 Vmax, Km=[S]

( small Km: high affinity of the enzyme for substrate; large Km: low affinity)

• Relationship of velocity to enzyme concentration:

The rate of the reaction is directly proportional to the enzyme concentrations.

• Order of the reaction:

When [S] << Km, the velocity is proportional to the

[S]---first order

When [S] >>Km, the velocity is constant and equal to

Vm---zero order

V= Vmax[S] / (Km+[S])

Figure 3.12 Michaelis-Menten kinetics. A plot of the reaction velocity as a function of the substrate concentration for an enzyme that obeys Michaelis-Menten kinetics.

• Lineweaver-Burke plot: If 1/Vo is plotted versus 1/[S], a straight line is

obtained (double-reciprocal plot). The plot is used to calculate Km and Vmax.

The equation: 1/Vo = Km/Vmax [S] + 1/Vmax

The intercept on the x axis is equal to -1/Km, The intercept on y axis is equal to 1/Vmax

(Michaelis-Menten equation: V0= Vmax[S] / (Km+[S]) )

Figure 3.13 A double-reciprocal or Lineweaver-Burk plot.

Ⅵ. Inhibition of enzyme activity

Inhibitors: substances that diminish the velocity

Reversible and irreversible inhibitionThe two most commonly encountered

types of reversible inhibition: competitive and non-competitive inhibitions

Figure 3.14 Distinction between a competitive and a noncompetitive inhibitor.

1. Competitive inhibition

Inhibitors binds to the same site that the substrate would occupy.

• Effect on Vmax : The inhibitor effect is reversed by

increasing [S]. The velocity reaches Vmax at a sufficiently high substrate concentration.

• Effect on Km : apparent Km increased, More substrate is needed to achieve ½ Vmax.

• Effect on Lineweaver-Burke plot: The plots of the inhibited and uninhibited

reactions intersect on the y axis at 1/Vmax

(Vmax is unchanged).

Figure 3.15 Reaction of a competitive inhibitor.

Figure 3.16 Kinetics of a competitive inhibitor. As the concentration of a competitive inhibitor increases, higher concentrations of substrate are required to attain a particular reaction velocity. The reaction pathway suggests how sufficiently high concentrations of substrate can completely relieve competitive inhibition.

Figure 3.17 Competitive inhibition illustrated on a double-reciprocal plot.

Figure 8.18 Enzyme inhibitors. The cofactor tetrahydrofolate and its structural analog methotrexate (MTX). Regions with structural differences are shown in red. MTX inhibits the dihydrofolate reductase, which plays a role in biosynthesis of purines and pyrimidines.

2. Non-competitive inhibition

• Inhibitor and substrate bind at different sites on the enzyme.

• Effect on Vmax : The inhibitor effect cannot be overcome by

increasing [S]. Thus, it will decrease Vmax of the reaction.

• Effect on Km : Km remains same• Effect on Lineweaver-Burke plot: Vmax decreases, Km is unchanged

Figure 3.19 Reaction of a non-competitive inhibitor.

Figure 3.19 Kinetics of a non-competitive inhibitor. The reaction pathway shows that the inhibitor binds both to free enzyme and to enzyme complex. Consequently, Vmax cannot be attained, even at high substrate concentrations.

Vmax

Figure 3.20 Noncompetitive inhibition illustrated on a double-reciprocal plot.

Irreversible inhibition

• Irreversible inhibitor tightly bound to the target enzyme either covalently or noncovalently. Some are important drugs.

• For example: penicillin can covalently modify the glycopeptide transpeptidase, preventing the synthesis of bacterial cell walls and thus killing the bacteria. Aspirin can covalently modify cyclooxygenase, reducing the synthesis of inflammatory signals.

Figure 3.21 Formation of a penicilloyl-enzyme complex. Penicillin reacts with transpeptidase to form an inactive complex, which is indefinitely stable.

Active site

Figure 3.22 Schematic representation of the peptidoglycan in Staphylococcus aureus. The sugars are shown in yellow, the tetrapeptides in red, and the pentaglycine bridges in blue. The cell wall is a single, enormous, bag-shaped macromolecule because of extensive cross-linking.

Ⅶ. Regulation of enzyme activityan organism is to coordinate its numerous metabolic processes

1. Allosteric regulation:

allosteric enzyme regulated by molecules called effectors (also modifiers)

no covalently binding at a site other than the active site

changing affinity of enzyme for its substrate

cooperativity– sigmoidal curve(V0 vs [S])

positive or negative effectors (feedback inhibition)

Effects of negative - or positive effectors on an allosteric enzyme.

A. Vmas is altered. B. The substrate concentration that gives half-maximal velocity(K0.5) is altered

2. Regulation of enzymes by covalent modification• Phosphorylation and dephosphorylation

protein kinase, adenosine triphosphate (ATP)

phosphatases• Response of enzyme to phosphorylation:

Phosphorylation may increase the activity (e.g. glycogen phosphorylase) or decrease the activity

(e.g. glycogen synthase) of an enzyme

3. Induction and repression of enzyme synthesis

• Regulation of the amount of enzyme present

altering the rate of enzyme synthesis

• Slow regulation (hours to days)

• For example: elevated levels of insulin cause an increase in the synthesis of key enzymes involved in glucose metabolism

Ⅷ. Enzymes in clinical diagnosis

1. Plasma enzymes: secreted by certain cell types (e.g. zymogen) and released from cells during normal cell turnover

healthy persons – constant level – steady state

Plasma is the fluid, noncellular part of blood. Serum is obtained by centrifugation of whole blood after its coagulation.

2. Alteration of plasma enzyme levels in disease states Tissue damage - release of enzymes into the plasma Diseases: heart, liver, skeletal muscle and other tissues3. Plasma enzymes as diagnostic tools Some enzymes in high activity in only one or a few tissues: reflecting damage to the corresponding tissue. e.g. Alanine aminotransferase (ALT) is abundant in liver. elevated level of ALT in plasma –hepatic damage

4. Isoenzymes and diseases of the heart

Most isoenzymes (isozymes) catalyze the same reaction with different primary structures. Different organs contain different isoenzymes. The pattern of isoenzymes found in plasma –the site of tissue damage

e.g. Creatine kinase (CK) and lactate dehydrogenase (LDH)

-myocardial infarction (when electrocardiogram is difficult to interpret)

• Quaternary structure of isoenzymes: containing different subunits in various combinations.

e.g. CK, dimer, two kinds of subunits (M and B);

three isoenzymes: CK1=BB, CK2=MB,

CK3=MM (CK2 increased in myocardial

infarction)

• Diagnosis of myocardial infarction: myocardial muscle is the only tissue that contains more than 5% of the total CK activity as the CK2 isoenzyme. CK2 in plasma appears 4 to 8 hours following onset of chest pain, and reaches a peak at 24 hours. LDH activity in plasma peaks 36 to 40 hours after infarction.

Figure 3.24 Subunit structure and electrophoretic mobility and enzyme activity of creatine kinase isoenzymes

Figure 3.25 Appearance of CK and LDH in plasma after a myocardial infarction

AC

TIV

ITY

CK2 activity in plasma peaks about 24 hours after infarction

LDH activity in plasma peaks about 36 to 40 hours after infarction

Summary Enzyme are protein catalysts that increase the

velocity of a chemical reaction by lowering the energy of the transition state. Enzymes are not consumed during the reaction they catalyze. Enzyme molecules contain a special pocket or cleft called the active site. The active site contains amino acid side chains that create a 3-D surface complementary to the substrate. The active site binds the substrate, forming an ES complex. ES is converted to enzyme-product (EP), which subsequently dissociates to enzyme and product.

An enzyme allows a reaction to proceed rapidly under conditions prevailing in the cell by providing an alternate reaction pathway with a lower free energy of activation. The enzyme does not change the free energies of the reactants or products and, therefore, does not change the equilibrium of the reaction. Most enzymes show Michaelis-Menten kinetics, and a plot of the initial reaction velocity, V0, against substrate concentration, [S], has a hyperbolic shape similar to the oxygen dissociation curve of myoglobin. Any substance that can diminish the velocity of such enzyme-catalyzed reactions is called an inhibitor.

The two most commonly encountered types of inhibition are competitive (which increases the apparent Km) and noncompetitive (which decreases the Vmax). In contrast, the multi-subunit allosteric enzymes frequently show a sigmoidal curve similar in shape to the oxygen dissociation curve of hemoglobin. They are frequently found catalyzing the committed (rate-limiting) step of a pathway. Allosteric enzymes are regulated by molecules called effectors that bind noncovalently at a site other than the active site. Effectors can be either positive or negative. An allosteric effector can alter the affinity of the enzyme for its substrate, or modify the maximal catalytic activity of the enzyme, or both.