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.. 10/28/2008. Biochemistry: Mechanisms. p. 3 of 56. Mechanism Topics. RegulationThermodynamicsEnzyme availabilityAllostery, revisited.
Enzyme Mechanisms and Regulation
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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, . . .
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:
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