11/5/2009biochem: specific mechanisms enzymes v: specific mechanisms; regulation andy howard...

11/5/2009Biochem: Specific Mechanisms

Enzymes V:Specific

Mechanisms; Regulation

Andy HowardIntroductory Biochemistry

10 November 2008

11/5/2009 Biochem: Specific Mechanisms P. 2 of 39

Examples of mechanisms

We’ll look at the serine protease mechanism in detail, and then explore a few other mechanisms to illustrate specific ideas

Then we’ll begin our discussion of regulation of enzymes


Mechanisms and Regulation Serine Proteases

Significance Catalytic residues Sequence of events Chymotrypsin Evolution

Other mechanisms Cysteinyl proteases

Lysozyme TIM

Regulation by thermodynamics

Enzyme availability Transcription Degradation Compartmentation

Allostery


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

NHCH

R1C

O

NH

CH

C

NH

R-1


Mechanism Active-site serine —OH …Without neighboring amino acids, it’s fairly non-reactive (naked ser-OH pKa ~ 14)

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 negative 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


What mechanistic concepts do serine proteases not illustrate? Quaternary structural effects(We’ll discuss this under regulation…)

Protein-protein interactions(Becoming increasingly important)

Allostery(also will be discussed under regulation)

Noncompetitive inhibition


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


Non-iClicker quiz, question 1

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-3-P (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 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)

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


Triosephosphate isomerase(TIM) dihydroxyacetone phosphate glyceraldehyde-3-phosphate

Km=10µMkcat=4000s-1

kcat/Km=4*108M-1s-1

DHAP

Glyc-3-P


TIM mechanism DHAP carbonyl H-bonds to neutral imidazole of his-95; proton moves from C1 to carboxylate of glu165

Enediolate intermediate (C—O- on C2) Imidazolate (negative!) form of his95 interacts with C1—O-H)

glu165 donates proton back to C2 See Fort’s treatment (http://chemistry.umeche.maine.edu/CHY431/Enzyme3.html)


Enzymes are under several levels of control

Some controls operate at the level of enzyme availability

Other controls are exerted by thermodynamics, inhibition, or allostery


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 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


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

11/5/2009biochem: specific mechanisms enzymes v: specific mechanisms; regulation andy howard...

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