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Enzyme Mechanisms and Regulation







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Enzyme Mechanisms and Regulation. Andy Howard Introductory Biochemistry, Fall 2008 Tuesday 28 October 2008. How do enzymes reduce activation energies?. We can illustrate mechanistic principles by looking at specific examples; we can also recognize enyzme regulation when we see it. Regulation
Enzyme Mechanisms and Regulation

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Slide 1

Enzyme Mechanisms and Regulation

Andy HowardIntroductory Biochemistry, Fall 2008Tuesday 28 October 2008

Biochemistry: Mechanisms

Slide 2

How do enzymes reduce activation energies?

  • We can illustrate mechanistic principles by looking at specific examples; we can also recognize enyzme regulation when we see it.

Biochemistry: Mechanisms

Slide 3

Regulation

Thermodynamics

Enzyme availability

Allostery, revisited

Mechanisms

Induced-fit

Tight Binding of Ionic Intermediates

Serine proteases

Other proteases

Lysozyme

Mechanism Topics

Biochemistry: Mechanisms

Slide 4

Examining enzyme mechanisms will help us understand catalysis

  • Examining general principles of catalytic activity and looking at specific cases will facilitate our appreciation of all enzymes.

Biochemistry: Mechanisms

Slide 5

Binding modes: proximity

William Jencks

  • We describe enzymatic mechanisms in terms of the binding modes of the substrates (or, more properly, the transition-state species) to the enzyme.

  • One of these involves the proximity effect, in which two (or more) substrates are directed down potential-energy gradients to positions where they are close to one another. Thus the enzyme is able to defeat the entropic difficulty of bringing substrates together.

Biochemistry: Mechanisms

Slide 6

Binding modes: efficient transition-state binding

  • Transition state fits even better (geometrically and electrostatically) in the active site than the substrate would. This improved fit lowers the energy of the transition-state system relative to the substrate.

  • Best competitive inhibitors of an enzyme are those that resemble the transition state rather than the substrate or product.

Biochemistry: Mechanisms

Slide 7

Proline racemase

  • Pyrrole-2-carboyxlate resembles planar transition state

Biochemistry: Mechanisms

Slide 8

Yeast aldolase

  • Phosphoglycolohydroxamate binds much like the transition state to the catalytic Zn2+

Biochemistry: Mechanisms

Slide 9

Adenosine deaminase with transition-state analog

  • Transition-state analog:Ki~10-8 * substrate Km

  • Wilson et al (1991) Science252: 1278

Biochemistry: Mechanisms

Slide 10

ADA transition-state analog

  • 1,6 hydrate of purine ribonucleoside binds with KI ~ 3*10-13 M

Biochemistry: Mechanisms

Slide 11

Induced fit

  • Refinement on original Emil Fischer lock-and-key notion:

  • both the substrate (or transition-state) and the enzyme have flexibility

  • Binding induces conformational changes

Biochemistry: Mechanisms

Slide 12

Example: hexokinase

  • Glucose + ATP  Glucose-6-P + ADP

  • Risk: unproductive reaction with water

  • Enzyme exists in open & closed forms

  • Glucose induces conversion to closed form; water can’t do that

  • Energy expended moving to closed form

Biochemistry: Mechanisms

Slide 13

Hexokinase structure

  • Diagram courtesy E. Marcotte, UT Austin

Biochemistry: Mechanisms

Slide 14

Tight binding of ionic intermediates

  • Quasi-stable ionic species strongly bound by ion-pair and H-bond interactions

  • Similar to notion that transition states are the most tightly bound species, but these are more stable

Biochemistry: Mechanisms

Slide 15

Serine protease mechanism

  • Only detailed mechanism that we’ll ask you to memorize

  • One of the first to be elucidated

  • Well studied structurally

  • Illustrates many other mechanisms

  • Instance of convergent and divergent evolution

Biochemistry: Mechanisms

Slide 16

The reaction

  • Hydrolytic cleavage of peptide bond

  • Enzyme usually works on esters too

  • Found in eukaryotic digestive enzymes and in bacterial systems

  • Widely-varying substrate specificities

    • Some proteases are highly specific for particular aas at position 1, 2, -1, . . .

    • Others are more promiscuous

CH

NH

C

NH

C

NH

R1

CH

O

R-1

Biochemistry: Mechanisms

Slide 17

Mechanism

  • Active-site serine —OH …Without neighboring amino acids, it’s fairly non-reactive

  • becomes powerful nucleophile because OH proton lies near unprotonated N of His

  • This N can abstract the hydrogen at near-neutral pH

  • Resulting + charge on His is stabilized by its proximity to a nearby carboxylate group on an aspartate side-chain.

Biochemistry: Mechanisms

Slide 18

Catalytic triad

  • The catalytic triad of asp, his, and ser is found in an approximately linear arrangement in all the serine proteases, all the way from non-specific, secreted bacterial proteases to highly regulated and highly specific mammalian proteases.

Biochemistry: Mechanisms

Slide 19

Diagram of first three steps

Biochemistry: Mechanisms

Slide 20

Diagram of last four steps

Diagrams courtesy University of Virginia

Biochemistry: Mechanisms

Slide 21

Chymotrypsin as example

  • Catalytic Ser is Ser195

  • Asp is 102, His is 57

  • Note symmetry of mechanism:steps read similarly L R and R  L

Diagram courtesy of Anthony Serianni, University of Notre Dame

Biochemistry: Mechanisms

Slide 22

Oxyanion hole

  • When his-57 accepts proton from Ser-195:it creates an R—O- ion on Ser sidechain

  • In reality the Ser O immediately becomes covalently bonded to substrate carbonyl carbon, moving - charge to the carbonyl O.

  • Oxyanion is on the substrate's oxygen

  • Oxyanion stabilized by additional interaction in addition to the protonated his 57:main-chain NH group from gly 193 H-bonds to oxygen atom (or ion) from the substrate,further stabilizing the ion.

Biochemistry: Mechanisms

Slide 23

Oxyanion hole cartoon

  • Cartoon courtesy Henry Jakubowski, College of St.Benedict / St.John’s University

Biochemistry: Mechanisms

Slide 24

Modes of catalysis in serine proteases

  • Proximity effect: gathering of reactants in steps 1 and 4

  • Acid-base catalysis at histidine in steps 2 and 4

  • Covalent catalysis on serine hydroxymethyl group in steps 2-5

  • So both chemical (acid-base & covalent) and binding modes (proximity & transition-state) are used in this mechanism

Biochemistry: Mechanisms

Slide 25

Specificity

  • Active site catalytic triad is nearly invariant for eukaryotic serine proteases

  • Remainder of cavity where reaction occurs varies significantly from protease to protease.

  • In chymotrypsin  hydrophobic pocket just upstream of the position where scissile bond sits

  • This accommodates large hydrophobic side chain like that of phe, and doesn’t comfortably accommodate hydrophilic or small side chain.

  • Thus specificity is conferred by the shape and electrostatic character of the site.

Biochemistry: Mechanisms

Slide 26

Chymotrypsin active site

  • Comfortably accommodates aromatics at S1 site

  • Differs from other mammalian serine proteases in specificity

Diagram courtesy School of Crystallography, Birkbeck College

Biochemistry: Mechanisms

Slide 27

Divergent evolution

  • Ancestral eukaryotic serine proteases presumably have differentiated into forms with different side-chain specificities

  • Chymotrypsin is substantially conserved within eukaryotes, but is distinctly different from elastase

Biochemistry: Mechanisms

Slide 28

iClicker quiz!

  • Why would the nonproductive hexokinase reaction H2O + ATP -> ADP + Pibe considered nonproductive?

  • (a) Because it needlessly soaks up water

  • (b) Because the enzyme undergoes a wasteful conformational change

  • (c) Because the energy in the high-energy phosphate bond is unavailable for other purposes

  • (d) Because ADP is poisonous

  • (e) None of the above

Biochemistry: Mechanisms

Slide 29

iClicker quiz, question 2:Why are proteases often synthesized as zymogens?

  • (a) Because the transcriptional machinery cannot function otherwise

  • (b) To prevent the enzyme from cleaving peptide bonds outside of its intended realm

  • (c) To exert control over the proteolytic reaction

  • (d) None of the above

Biochemistry: Mechanisms

Slide 30

Question 3: what would bind tightest in the TIM active site?

  • (a) DHAP (substrate)

  • (b) D-glyceraldehyde (product)

  • (c) 2-phosphoglycolate(Transition-state analog)

  • (d) They would all bind equally well

Biochemistry: Mechanisms

Slide 31

Convergent evolution

  • Reappearance of ser-his-asp triad in unrelated settings

  • Subtilisin: externals very different from mammalian serine proteases; triad same

Biochemistry: Mechanisms

Slide 32

Subtilisin mutagenesis

  • Substitutions for any of the amino acids in the catalytic triad has disastrous effects on the catalytic activity, as measured by kcat.

  • Km affected only slightly, since the structure of the binding pocket is not altered very much by conservative mutations.

  • An interesting (and somewhat non-intuitive) result is that even these "broken" enzymes still catalyze the hydrolysis of some test substrates at much higher rates than buffer alone would provide. I would encourage you to think about why that might be true.

Biochemistry: Mechanisms

Slide 33

Cysteinyl proteases

  • Ancestrally related to ser proteases?

  • Cathepsins, caspases, papain

  • Contrasts:

    • Cys —SH is more basicthan ser —OH

    • Residue is less hydrophilic

    • S- is a weaker nucleophile than O-

Diagram courtesy ofMariusz Jaskolski,U. Poznan

Biochemistry: Mechanisms

Slide 34

Papain active site

Diagram courtesy Martin Harrison,Manchester University

Biochemistry: Mechanisms

Slide 35

Hen egg-white lysozyme

  • Antibacterial protectant ofgrowing chick embryo

  • Hydrolyzes bacterial cell-wall peptidoglycans

  • “hydrogen atom of structural biology”

    • Commercially available in pure form

    • Easy to crystallize and do structure work

    • Available in multiple crystal forms

  • Mechanism is surprisingly complex (14.7)

HEWLPDB 2vb10.65Å15 kDa

Biochemistry: Mechanisms

Slide 36

Mechanism of lysozyme

  • Strain-induced destabilization of substrate makes the substrate look more like the transition state

  • Long arguments about the nature of the intermediates

  • Accepted answer: covalent intermediate between D52 and glycosyl C1 (14.39B)

Biochemistry: Mechanisms

Slide 37

The controversy

Biochemistry: Mechanisms

Slide 38

Regulation of enzymes

  • The very catalytic proficiency for which enzymes have evolved means that their activity must not be allowed to run amok

  • Activity is regulated in many ways:

    • Thermodynamics

    • Enzyme availability

    • Allostery

    • Post-translational modification

    • Protein-protein interactions

Biochemistry: Mechanisms

Slide 39

Thermodynamics as a regulatory force

  • Remember that Go’ is not the determiner of spontaneity: G is.

  • Therefore: local product and substrate concentrations determine whether the enzyme is catalyzing reversible reactions to the left or to the right

  • Rule of thumb: Go’ < -20 kJ mol-1 is irreversible

Biochemistry: Mechanisms

Slide 40

Enzyme availability

  • The enzyme has to be where the reactants are in order for it to act

  • Even a highly proficient enzyme has to have a nonzero concentration

  • How can the cell control [E]tot?

    • Transcription (and translation)

    • Protein processing (degradation)

    • Compartmentalization

Biochemistry: Mechanisms

Slide 41

Transcriptional control

  • mRNAs have short lifetimes

  • Therefore once a protein is degraded, it will be replaced and available only if new transcriptional activity for that protein occurs

  •  Many types of transcriptional effectors

    • Proteins can bind to their own gene

    • Small molecules can bind to gene

    • Promoters can be turned on or off

Biochemistry: Mechanisms

Slide 42

Protein degradation

  • All proteins havefinite half-lives;

  • Enzymes’ lifetimes often shorter than structural or transport proteins

  • Degraded by slings & arrows of outrageous fortune; or

  • Activity of the proteasome, a molecular machine that tags proteins for degradation and then accomplishes it

Biochemistry: Mechanisms

Slide 43

Compartmentalization

  • If the enzyme is in one compartment and the substrate in another, it won’t catalyze anything

  • Several mitochondrial catabolic enzyme act on substrates produced in the cytoplasm; these require elaborate transport mechanisms to move them in

  • Therefore, control of the transporters confers control over the enzymatic system

Biochemistry: Mechanisms

Slide 44

Allostery

  • Remember we defined this as an effect on protein activity in which binding of a ligand to a protein induces a conformational change that modifies the protein’s activity

  • Ligand may be the same molecule as the substrate or it may be a different one

  • Ligand may bind to the same subunit or a different one

  • These effects happen to non-enzymatic proteins as well as enzymes

Biochemistry: Mechanisms

Slide 45

Substrates as allosteric effectors (homotropic)

  • Standard example: binding of O2 to one subunit of tetrameric hemoglobin induces conformational change that facilitates binding of 2nd (& 3rd & 4th) O2’s

  • So the first oxygen is an allosteric effector of the activity in the other subunits

  • Effect can be inhibitory or accelerative

Biochemistry: Mechanisms

Slide 46

Other allosteric effectors (heterotropic)

  • Covalent modification of an enzyme by phosphate or other PTM molecules can turn it on or off

  • Usually catabolic enzymes are stimulated by phosphorylation and anabolic enzymes are turned off, but not always

  • Phosphatases catalyze dephosphorylation; these have the opposite effects

Biochemistry: Mechanisms

Slide 47

Cyclic AMP-dependent protein kinases

  • Enzymes phosphorylate proteins with S or T within sequence R(R/K)X(S*/T*)

  • Intrasteric control:regulatory subunit or domain has a sequence that looks like the target sequence; this binds and inactivates the kinase’s catalytic subunit

  • When regulatory subunits binds cAMP, it releases from the catalytic subunit so it can do its thing

Biochemistry: Mechanisms

Slide 48

Kinetics of allosteric enzymes

  • Generally these don’t obey Michaelis-Menten kinetics

  • Homotropic positive effectors produce sigmoidal (S-shaped) kinetics curves rather than hyperbolae

  • This reflects the fact that the binding of the first substrate accelerates binding of second and later ones

Biochemistry: Mechanisms

Slide 49

T  R State transitions

  • Many allosteric effectors influence the equilibrium between two conformations

  • One is typically more rigid and inactive, the other is more flexible and active

  • The rigid one is typically called the “tight” or “T” state; the flexible one is called the “relaxed” or “R” state

  • Allosteric effectors shift the equilibrium toward R or toward T

Biochemistry: Mechanisms


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