Enzyme Mechanisms and Regulation
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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Enzyme Mechanisms and Regulation
Andy HowardIntroductory Biochemistry, Fall 2008Tuesday 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.
Tight Binding of Ionic Intermediates
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.
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.
- Pyrrole-2-carboyxlate resembles planar transition state
- 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) Science252: 1278
ADA transition-state analog
- 1,6 hydrate of purine ribonucleoside binds with KI ~ 3*10-13 M
- Refinement on original Emil Fischer lock-and-key notion:
- both the substrate (or transition-state) and the enzyme have flexibility
- Binding induces conformational changes
- 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
- 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
- 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
- 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.
- 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
- 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
- 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
- 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
- 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
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
- Reappearance of ser-his-asp triad in unrelated settings
- Subtilisin: externals very different from mammalian serine proteases; triad same
- 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.
- Ancestrally related to ser proteases?
- Cathepsins, caspases, papain
- 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)
HEWLPDB 2vb10.65Å15 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)
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:
- Enzyme availability
- 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
- 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)
- 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
- 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
- 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
- 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