enyzymes and how they work

99:163 Biochemistry, Shea

99:163 Medical BiochemistryEnzymes and How They Work

with emphasis on ProteasesLectures 8 & 9

Instructor: Dr. Madeline A. Shea, Prof. of Biochemistry4-450 BSB, 335-7885, [email protected]

Reminders:Help is available to prepare for Exam #1 - Thursday, Sept. 10

TA Discussion Sections - Tuesday, 7 pm, 2189 MERF

Dr. Shea’s Optional “Open Group” Review SessionThis Friday, 9/4, 2:30 to 4:30 pm - Spivey Auditorium

2

Basic Properties of Enzymes

I. Outline and Study QuestionsII. Scientific Topics

A. Reading ListB. Enzymes

i. Overviewii. Rate Enhancementsiii. Kinetics (rates) vs. Thermodynamics (Free Energy, ∆G)iv. Specificityv. Example Chymotrypsin (Protease)

C. Enzyme Inhibition: Competitive & NoncompetitiveD. HIV ProteaseE. ZymogensF. Allosteric Enzyme: ATCase

III. Reading - all Biochemistry textbooks cover enzymesLehninger 4e Ch. 6, Lippincott Ch. 5Berg, Tymoczko, Stryer. Biochemistry5e: Ch. 8, 9 and 10Devlin. Biochemistry Ch. 4


3

Relevant §’s in Stryer5e

8.1 Enzymes are Powerful and Highly Specific Catalysts

8.2 Free Energy is a Useful Thermodynamic Function for Understanding Enzymes

8.3 Enzymes Accelerate Reactions by Facilitating Formation of Transition State

8.4. Michaelis-Menten Model accounts for Kinetic Properties of Many Enzymes

8.5 Enzymes can be Inhibited by Specific Molecules

8.6 Vitamins and Co-enzymes

9.1 Proteases

10.1 Aspartate Transcarbamoylase is Allosterically Inhibited by the End-Product of its Pathway

10.3 Isozymes Provide a Means of Regulation Specific to Distinct Tissues and Developmental Stages

10.4 Covalent Modification is Means of Regulating Enzyme Activity

10.5 Many Enzymes are Activated by Specific Proteolytic Cleavage

4

Study Guide

1. Indicate whether each of the following is true or false.If the statement is false, explain why.

a. The rate of an enzyme-catalyzed reaction is linearly proportional to thesubstrate concentration.

b. An enzyme-catalyzed reaction velocity reaches Vmax when thesubstrate concentration is equal to twice Km.

c. The Michaelis constant (Km) of an enzyme identifies the substrateconcentration at which an average of 50% of the active sites of theenzyme have substrate bound to them.

d. Enzymes lower the free energy difference between reactants andproducts.

2. Explain differences between the “Lock and Key” and “InducedFit” models of substrate binding.

3. What are competitive and non-competitive enzyme inhibition?What types of effects do inhibitors have on substrate binding?How do they affect the rate of conversion of substrate to product?

4. What is a zymogen? what is one of their biological roles?5. Explain how rising [CTP] has an effect on the allosteric regulation

of aspartate transcarbamoylase (ATCase).


5

Enzymes are Biological Catalysts of ReactionsSubstrate(s) k Product(s)

Catalysts accelerate reactions to work at 1 atm., 37˚CSpeed up reactions by many orders of magnitude (see table).Protein enzymes often have names ending in -ase

Examples: nuclease, polymerase, kinase, phosphataseRNA-enzymes are called ribozymes.

Catalysts are NOT consumed in the reactionimportant for cascade processes (not used up in the reaction)available for re-use without re-synthesis

Most enzymes are highly specific (bind few substrates)Proteases usually cleave after a specific type of amino acid.Trypsin cleaves after lysine and arginine (both basic AA)

but not after other amino acids.Enzymes can be inhibited and regulated.

Multiple classes of inhibitors: binding at the active site vs. afar.Some enzymes are multi-subunit & have allosteric regulation.

6

Example of Membrane-Spanning EnzymeCa2+-ATPase: Hydrolyzes ATP & Pumps Ca2+

Energy in the form of ATP is required to pump calcium againsta chemical gradient. So, ATP is hydrolyzed concomitant withcalcium pumping. But, most enzymes do not require ATP.


7

Soluble Metabolic Enzyme: Hexokinase

2yhx.pdb

1hjk.pdbred = D-glucose

Induced Fit - crescent-shaped enzyme closes around substrate, S

8

Rate Enhancement by EnzymesExamples show Increases of 106 to 1017

Numbers provided only to demonstrate known range.Students are not responsible for memorizing this list.

Half-life: time needed for half of starting material to transform from reactant to product


9

Enzymes Vary in Turnover Number*, andpH of Optimal Catalysis

* Catalyzed reactions per second.

10

Six Major Classes of Enzymes


11

Families of Proteases

12

Enzymes move Electrons, Atoms or Functional Groupsusing Sidechains in Active Sites

Lehninger


13

Inorganic Elements - Enzyme Cofactors

Lehninger

14

Coenzymes = Transient Carriers

Lehninger


15

Enzymes, Substrates & Products

E + S k ES (k EP ) k E + P

V0Initial Rate of

Product Releasefrom Enzyme

Stryer5e

16

Legend Enzymes, Substrates & Products

E + S k ES k E + PIn an enzyme-catalyzed reaction, the substrates (S) (the

molecule that will be modified) binds reversibly to theactive site of the enzyme (E).

This temporary association causes a reduction in the energyrequired to “activate” the reaction of the substratemolecule (called the “activation energy”) so that theproducts (P) of the reaction are formed.

It is possible to measure the formation of product, or the lossof substrate, from the moment the reactants are broughttogether until the reaction has stopped.

The amount of product formed is measured at regularintervals and the rate is plotted on a graph vs. time, like thatabove. Initial velocity measurements from many such plotsof Product vs. time are combined into one plot of V vs. [S](examples later in slides).


17

Enzymes cannot alter equilibrium position or ∆G˚

Reaction Rate is not proportional to “Thermodynamic Spontaneity”Glucose + 6 O2 ok 6 CO2 + 6 H2O -689 kcal/mol

but sugar is stable on the shelf for years …

A + B C + D

Standard Free Energy = ∆G˚= -RTln(Keq) ∆G˚ = Energy of products - Energy of reactantsThermodynamic spontaneity depends on ∆G˚ being < 0.∆G˚ independent of path, does not predict ratesRate depends on energy of activation, unrelated to ∆G˚

Chemical ReactionsKinetics vs. Thermodynamics

!

Keq =[C]eq • [D]eq[A]eq • [B]eq

18

Free Energy vs. Reaction ProgressEnzymes Lower Activation Energy, ∆G†

Reactants k Transition State k ProductsA + B k A' + B' k C + D

S k S' k P∆G† = Energy to initiate the reaction.

= ∆Gtransition state - ∆G substrateThe chemical properties of transitionstates are difficult to determine! But,understanding the transition statemeans understanding the mechanism.

Enzymes lower the activation energyThis increases the rate at which thebarrier is climbed (i.e., likelihood itwill be reached). But, an enzyme lowersthe barrier for crossing in the otherdirection, too.

Enzyme does not changeequilibrium concentrations

Stryer5e Fig. 8.3Enzyme DecreasesActivation Energy


19

Correspondence between Keq and ∆G

Lehninger Slide is provided for reference. Students need not memorize #’s.

20

Catalysis Requires that Enzyme beComplementary to transition State

Lehninger


21

Formation of ES (enzyme-substrate) Complex

Enzyme + Substrate k E•S k E + ProductConsider a single substrate (S) and a single product (P).

The principles are the same for multiple S’s, P’s.Enzyme binds substrate at active site

Key Features of Active SitesSmall fraction of total volume of enzymeActive site is a cleft or crevice or interface -

partly to control water and access to cofactors.3-D configuration of active site:

residues far apart in sequence may contribute to siteSubstrate is bound by multiple, weak interactionsSpecific shape - counter-productive for substrate to bind “too well” -

otherwise it will not be converted to product.

Stryer5e Fig. 8.5Cytochrome P-450 binding Camphor

22

“Active Site Residues” May beClose in 3˚ but Distant in 1˚Structure

Hen Egg-White LysozymeDegrades some bacterial cell wallsSee “Living Figure” on Stryer5e website.

Stryer5e Fig. 8.7


23

Substrate Binding to Active Site:2 Common Models of Specificity

“Lock & Key”Assumes high degree ofsimilarity between shape ofsubstrate and bindingpocket on the enzyme.S fits like a key in a lock.

“Induced Fit”Accounts for enzyme flexibility -binding of S induces change inconformation in E.Binding site shape of enzyme alonediffers from ES complex.

Stryer5e

24

Reaction Catalyzed by Chymotrypsin: Cleavage of peptide bond

Cleavage of peptidebond after aromaticor large hydrophobicresidues. Scissile(susceptible) bondsindicated by arrows.Fig. 9.1/Stryer5e

Active site containsunusually reactive

Serine (@195)(28 total Ser in chymotrypsin).

Fig. 9.2/Stryer5e

Lehninger Fig 6.21: Chymotrypsin Mechanism http://bcs.whfreeman.com/lehninger/


25

Key Properties of Transition States

Proximity &

Orientation

When transition state is formed, atoms that need toreact are brought close to together. Also, they havefew choices of how to interact. E is designed toproduce a single P! (limited side reactions)

Unique Mode of

Catalysis

Many proteins may do the "same" job - such ashydrolyze proteins by adding water across the peptidebond. However the properties of the active sitedetermine the which peptide bonds will be broken.

Chymotrypsin Stryer5e Fig. 9.10

Stryer5e Fig. 9.13

LysArg

AlaSer

Phe,Tyr,Trp, Met

26

Tertiary Structure SimilaritiesTrypsin (blue) vs. Chymotrypsin (red)

Only Cα carbons are shown (i.e., backbone carbons).Average deviation in position of backbone is only 1.7 Å.But chymotrypsin cuts after hydrophobic side chains

while trypsin after basic/alkaline residues.

Stryer5e Fig. 9.12


27

Enzyme distorts S and Creates P(Chymotrypsin cuts Peptide Bond)

!

E + S k1 "# k$1

ES k2 " E + P S

P ✄

Rate of Product formation will plateauwhen all scissors are snipping

(analogous to all E saturated with S).

28

Rates of Proteolysis by ChymotrypsinCatalytic Properties depend on Active Site Chemistry

Structure of chymotrypsin (Fig. 9.10/Stryer5e) showing the active site:Serine 195 H-bonded to Histidine 57, with Aspartate 102 completingthe “catalytic” triad. Vo, initial rate of hydrolysis, determined forcleavage of “S”* monitoring appearance of yellow product.* N-acetyl-L-phenylalanine p-nitrophenylester (see Fig. 9.3, Stryer5e)

!

E + S k1 "# k$1

ES k2 " E + P


29

Definitions of Terms

k1 = formation of ES complex

k-1 = dissociation of ES complex to form free E & S

k2 = conversion of ES to P and release of free E

!

E + Sk1 "# k$1

ESk2 " E + P

Remember that catalyst (free enzyme) must be regenerated atend of each “turnover” or conversion of S to P.

• Mechanism for an enzyme-catalyzed reaction with no "backreaction" (P converted to S). Thus, no rate k-2 specified.

• Rates k-1 and k1 describe equilibrium of E and S. Intermediateproduct must be formed and final product must be released.

• As long as [P] is low (which is usually the case in biologicalreactions because it’s being made to be used in a subsequentreaction), the conversion of ES to P is irreversible.

30

Changes in Concentrations ofE, S, ES, & P over course of time

Stryer5e Fig. 8.13

!

E + S k1 "# k$1

ES k2 " E + P

“Steady State” conditions apply whenRate of formation = Rate of breakdown

*

*


31

Vo vs. [Substrate]Plot of initial* velocity vs. [S](*reverse reaction is negligible).Stryer5e Fig. 8.11

Curve is hyperbolic.Substrate binding to Enzyme is“same” as O2 binding to myoglobin!

!

E + S k1 "# k$1

ES k2 " E + P

Vo = k2 • [ES]

At high [S], rate is constantbecause all E is in ES complex.

Vmax = k2 • [E]total

At Steady State,formation = breakdown of ES

[E] [S] / [ES] = (k-1 + k2 )/k1 = Km

Km = “Michaelis Constant”

!

Vo = k2[E]Total[S]

[S]+ Km

Vo = Vmax

1Km[S]

1Km[S]+ 1

= VmaxK'[S]K'[S]+ 1

32

Vo vs. [Substrate]

!

E + S k1 "# k$1

ES k2 " E + P

[E] [S] / [ES] = (k-1 + k2 )/k1 = Km

Km = “Michaelis Constant”

1/Km = K’

Limiting Case

If k-1 >> k2 : it is more likelyfor ES to dissociate than to makeproduct

Km = k-1/k1 = Kdssn

!

Vo = VmaxK'[S]1+ K'[S]

Lehninger Enzyme Kinetics Simulation, Ch. 6 http://bcs.whfreeman.com/lehninger/


33

Enzyme Km Values: Like Kdssn (molar units)Indicate Concentration Range of Activity

Numbers provided only to demonstrate known range.Students are not responsible for memorizing this list.

34

Stryer5e Web Tutorials Recommendedhttp://bcs.whfreeman.com/stryer/cat_010/ch08/ch08xa01a.htmhttp://bcs.whfreeman.com/stryer/cat_010/ch09/ch09xa01a.htm

Demonstrations & Simulationsof Chymotrypsin (Protease)Digestion (or Hydrolysis) ofChromogenic (color-giving)Substrate


35

Addendum on Chymotrypsin Hydrolysis

• Catalysis reaction proceeds through covalent intermediate(“Acyl-Enzyme”) at Ser195.

• Peptide with free Amino Group is released first.• Peptide with free Carboxyl group is released second.• Ser OH is regenerated after 2nd product released.

Released Released

Ser195

36

In Catalytic Triad: His57 + Ser195 =Protonated Histidine + Alkoxide Ion Serine

• H-bond Network between Asp102, His57 and Ser195separates charge between Serine and Histidine:His57 accepts a proton (acts as a base), whileSer195 donates a proton (acts as an acid).

• Resulting Alkoxide ion is potent nucleophile,which attacks the peptide bond …


37

Chymotrypsin: Multi-step Mechanismto Cleave a Peptide Bond

“S” =Peptide

Active Site

Free Amino

Free Carboxyl

1

2

Lehninger Fig 6.21: Chymotrypsin Mechanism http://bcs.whfreeman.com/lehninger/

38

Chymotrypsin Holds Intermediate with H-bonds

Fig. 9.9Tertiary structure of the

active site stabilizes thetetrahedral intermediateformed during catalysis.

H-bonds from NH groups ofEnzyme hold Substrate inplace.

Gly193 fits well in the activesite, leaving room for S.


39

Double-Reciprocal Plot of Enzyme KineticsPlease see Stryer5e, Ch. 8 Appendix

Stryer5e Fig. 8.36

Disclaimer:Necessary in days of straight-edge and graph paper …but better (and free) computerized approaches exist now.No graphs of this kind will be on Exam #1.

40

Enzymes are Inhibited & Regulated

Conserve massDon’t break down "S" farther than needed.

Conserve Energytake little steps so that each is “manageable”.

Coordinate PathwaysSome intermediates are used in other pathwaysor serve as regulatory molecules in others.

There are multiple mechanisms for this. Enzymes mayintegrate chemical “input” from many sources throughdiffusion/binding of substrates and effectors. Catabolism& Metabolism generally proceed in multiple steps. Eachhas its own enzyme. What is advantage of this?


41

Comparison of Inhibition Mechanisms”Competitive” vs. “Noncompetitive”

S: “Hey, get outof my chair!”

S: “My chair has changed! I don’t

fit as well”

Stryer5e

42

Action of Competitive Inhibitor

Possible to “swamp out” acompetitive inhibitor withincreasing levels ofsubstrate because theycompete for same site.

The maximal relative rate*is not affected, but Km is.

Recall: Km = [S] at half-maximal rate or Vmax/2.

Stryer5e Fig. 8.17

*


43

Appearance of Competitive Inhibitionon a Double-Reciprocal Plot

Stryer5e Fig. 8.36

Slopes are different.

Curves cross at singleintercept on 1/V axis.

Note: Students will notbe tested by Dr. Shea onthe shapes of doublereciprocal plots.

44

Action of a Noncompetitive Inhibitor

Cannot “swamp out”a noncompetitiveinhibitor withincreasing levels ofsubstrate.

The maximal relativerate is affected,but not the Km,the [S] at themidpoint of thecurve (Vmax/2).

Stryer5e Fig. 8.18


45

Appearance of Noncompetitive Inhibitionon a Double-Reciprocal Plot

Stryer5e Fig. 8.38

Slopes are different.

Curves cross at singleintercept on 1/[S] axis.

Note: Students will notbe tested by Dr. Shea onthe shapes of the doublereciprocal plots.

46

HIV Protease: Drug Target

HIV proteaseis essential for infectivityclips immature forms of

viral proteins to makethem active.

is an aspartyl protease(i.e., Asp is in active site)

is homodimer (symmetric)

Stryer5e Fig. 9.19

Aspartyl groups (one fromeach monomer in dimer)

interact with Crixivan. Flapshave closed on Inhibitor.

Challenge is finding drug thatbinds selectively to single E.

Stryer5e Fig. 9.21


47

Crixivan: Inhibitor of HIV Protease

Inhibitor mimics the natural peptide substrate: chemical groups are available to mimic amino acids that would fit into the recognition sites on the enzyme.

Stryer5e Fig. 9.21Stryer5e Fig. 9.20

48

Zymogens:Benign Precursors to Destructive Enzymes

• Processing of a Precursor Protein– Zymogen is an inactive precursor that can become an active enzyme by

changes in covalent structure - usually proteolytic cleavage or removal ofpart of the enzyme. Usually an “irreversible” step - very unlikely to reverse.

• Chymotrypsinogen– Precursor to chymotrypsin. 5 S-S linkagesin

245 AA’s. Mixed β-sheet + some α helix.– 2 clips, 3 fragments bonded by Cys-Cys.

– Removal of a few amino acids changestertiary structure and changes interactionsnecessary for active site conformation.

• Blood Clotting Cascade– Fibrinogen (soluble) > Fibrin (insoluble) converted by the action of

thrombin on fibrinogen. To control the level of thrombin it exists asprecursor, prothrombin. Lack of clotting factors > hemophilia

1 245

245

1kdq.pdb


49

Processing of Chymotrypsin

50

Example of Negative Feedback RegulationProkaryotic Biosynthesis of Pyrimidines

CTP is (1) end-product of pathway and (2) negative feedback regulator

ATCase: Aspartyl Transcarbamoylase

Stryer5e


51

ATCase: Aspartyl Transcarbamoylasec6r6 (2c3 + 3r2 )

A. Top View

B. Side View

Stryer5e Fig. 10.5

52

ATCase: 2 Trimers of “c” catalytic subunits

Stryer5e Fig. 10.7


53

T-to-R Quaternary Transition

Stryer5e Fig. 9.21

54

ATCase: Multi-Subunit Enzymewith Allosteric Regulators

Reaction depends on [S] butcurve is sigmoidal with an apparent "lag”.

Feedback inhibition of control pointMajor role - both midpoint and shape regulated by allosteric effectors

Stryer5e Fig. 10.11 Stryer5e Fig. 10.14ATP stabilizes the R (active) quaternary state

Stryer5e Fig. 10.15CTP inhibits catalysisby stabilizing the T state

Back to: NIGMS Home > Featured Areas > Pharmacogenetics ResearchNetwork > Background Information

Search:

Your genes determine a lot about how youlook. They also play a key role in how yourbody responds to medicines.

EVERYONE RESPONDS DIFFERENTLY TO MEDICINES.The dose of a drug that curesone person can be ineffective—or even toxic—in someone else. The reason? Genes(although a person's age, weight, lifestyle, and other medicines also play a role).

By understanding the genetic basis of drug responses, scientists hope to enable doctors toprescribe the drugs and doses best suited for each individual.

Scientists studying how genes affect responses to drugs are engaged in an active field of research known as pharmacogenetics orpharmacogenomics. (These terms are often used interchangeably, although to scientists they can have subtly different meanings.)

These researchers focus on variations in the protein molecules that interact with medicines moving through the body. Variations in theseprotein molecules are largely responsible for individual differences in drug responses.

THE ANTICIPATED BENEFITS OF PHARMACOGENETICS RESEARCH INCLUDE:

More Accurate DosingInstead of basing a starting dose on characteristics like weight and age, doctors may use a patient’s genetic profile to predict how well his orher body will handle a medicine. Then, doctors could adjust the dose accordingly.

New, More Targeted DrugsPharmaceutical companies would be able to develop and market drugs for people with specific genetic profiles. Testing a drug only in thoselikely to benefit from it could streamline clinical trials and speed the process of getting a drug to market.

Improved Health CareIn the future, doctors may be able to prescribe the right dose of the right medicine the first time for everyone. This would mean that patientsreceive medicines that are safer and more effective for them, speeding recovery and reducing adverse drug reactions (estimated at 100,000deaths and 2 million hospitalizations annually in the United States1). In this way, taking individual genetic profiles into account whendeveloping and prescribing medicines would lead to better health care overall.

ALREADY IN USE:

For a few medications, doctors are already starting to use pharmacogenomic information. For example, some research hospitals routinelyexamine groups of genes in children with leukemia before treating them. Different versions of these genes can result in dramatically differentresponses to antileukemia treatments. Based on the results of these genetic tests, doctors can prescribe the safest and most effective drugregimen for each child.

In addition, the U.S. Food and Drug Administration has started to modify the labels of some medicines to include pharmacogenomicinformation. This ensures that the drugs are as safe as possible and helps doctors customize doses for individual patients. Examples ofdrugs whose labels have changed are irinotecan (Camptosar®), used to treat colorectal cancer; mercaptopurine (Purinethol®), used to treatinflammatory bowel disease and childhood leukemia; and warfarin (Coumadin®), a blood-thinner used to prevent strokes.

NIH NETWORK LEADS THE WAY:

To foster an organized, large-scale effort in pharmacogenomics, the National Institute of General Medical Sciences (NIGMS), together withother components of the National Institutes of Health (NIH), formed the Pharmacogenetics Research Network (PGRN). This nationwidecollaboration of hundreds of scientists study genes and medicines relevant to a wide range of diseases, including asthma, depression,cancer, and heart disease. They share their findings in a knowledge base available to all scientists (http://www.pharmgkb.org/).

NIGMS also supports research on the ethical, legal, and social implications of the use of pharmacogenetic information and works closelywith associations and task forces on these issues.

In 2008, PGRN scientists joined with Japanese scientists to form a Global Alliance for Pharmacogenomics.

The NIH Pharmacogenetics Research Network has made many advances in understanding the way genes affect individualresponses to medicines.

More Information

Educational booklet on personalized medicines, Medicines for You

Pharmacogenetics Research Network

National Center for Biotechnology Information, A Science Primer: The Promise of Pharmacogenomics

Department of Health and Human Services, HIPAA information page

National Human Genome Research Institute, Human Genome Information pageInternational HapMap

U.S. Food and Drug Administration, How FDA Advances Personalized Medicine

Contact

If you would like more information on pharmacogenomic research supported by the National Institute of General Medical Sciences, pleasecontact Alisa Machalek at 301-496-7301 or [email protected].

About NIGMS

NIGMS supports basic biomedical research that is the foundation for advances in the diagnosis, treatment, and prevention of disease.NIGMS is part of the National Institutes of Health, U.S. Department of Health and Human Services.

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Personalized Medicines Fact Sheet - National Institute of General Medical Sciences http://www.nigms.nih.gov/Initiatives/PGRN/Background/FactSheet.htm

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