10/28/2008 biochemistry: mechanisms 1 enzyme mechanisms and regulation andy howard introductory...

10/28/2008Biochemistry: Mechanisms 1

Enzyme Mechanisms and Regulation

Andy HowardIntroductory Biochemistry, Fall 2008

Tuesday 28 October 2008

10/28/2008 Biochemistry: Mechanisms p. 2 of 56

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.


Mechanism Topics

Regulation Thermodynamics Enzyme availability Allostery, revisited

Mechanisms Induced-fit Tight Binding of

Ionic Intermediates

Serine proteases Other proteases Lysozyme


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.


Binding modes: proximity

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.

William Jencks


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.


Proline racemase

Pyrrole-2-carboyxlate resembles planar transition state


Yeast aldolase

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


Adenosine deaminase with transition-state analog Transition-state analog:

Ki~10-8 * substrate Km

Wilson et al (1991) Science 252: 1278

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.


ADA transition-state analog 1,6 hydrate of

purine ribonucleoside binds with KI ~ 3*10-13 M


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


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


Hexokinase structure Diagram courtesy E. Marcotte, UT Austin


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


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


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

NH

CH

R1C

O

NH

CH

C

NH

R-1


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.


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.


Diagram of first three steps


Diagram of last four steps

Diagrams courtesy University of Virginia


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


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.


Oxyanion hole cartoon

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


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


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.


Chymotrypsin active site Comfortably

accommodates aromatics at S1 site

Differs from other mammalian serine proteases in specificity

Diagram courtesy School of Crystallography, Birkbeck College


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


iClicker quiz! Why would the nonproductive hexokinase

reaction H2O + ATP -> ADP + Pi

be 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


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


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


Convergent evolution Reappearance of ser-his-asp triad in

unrelated settings Subtilisin: externals very different from

mammalian serine proteases; triad same


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.


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


Papain active site

Diagram courtesy Martin Harrison,Manchester University


Hen egg-white lysozyme Antibacterial protectant of

growing 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)

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

HEWLPDB 2vb1

0.65Å15 kDa


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)


The controversy


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


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


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


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


Protein degradation All proteins have

finite 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


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


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


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


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


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


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


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

10/28/2008 biochemistry: mechanisms 1 enzyme mechanisms and regulation andy howard introductory...

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