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11/5/2009Biochem: Specific Mechanisms Enzymes V: Specific Mechanisms; Regulation Andy Howard...
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Transcript of 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
11/5/2009 Biochem: Specific Mechanisms P. 3 of 39
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
11/5/2009 Biochem: Specific Mechanisms P. 4 of 39
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
11/5/2009 Biochem: Specific Mechanisms P. 5 of 39
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
11/5/2009 Biochem: Specific Mechanisms P. 6 of 39
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.
11/5/2009 Biochem: Specific Mechanisms P. 7 of 39
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.
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Diagram of first three steps
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Diagram of last four steps
Diagrams courtesy University of Virginia
11/5/2009 Biochem: Specific Mechanisms P. 10 of 39
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
11/5/2009 Biochem: Specific Mechanisms P. 11 of 39
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.
11/5/2009 Biochem: Specific Mechanisms P. 12 of 39
Oxyanion hole cartoon
Cartoon courtesy Henry Jakubowski, College of St.Benedict / St.John’s University
11/5/2009 Biochem: Specific Mechanisms P. 13 of 39
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
11/5/2009 Biochem: Specific Mechanisms P. 14 of 39
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
11/5/2009 Biochem: Specific Mechanisms P. 15 of 39
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.
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Chymotrypsin active site Comfortably accommodates aromatics at S1 site
Differs from other mammalian serine proteases in specificity
Diagram courtesy School of Crystallography, Birkbeck College
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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
11/5/2009 Biochem: Specific Mechanisms P. 18 of 39
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
11/5/2009 Biochem: Specific Mechanisms P. 19 of 39
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
11/5/2009 Biochem: Specific Mechanisms P. 20 of 39
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
11/5/2009 Biochem: Specific Mechanisms P. 21 of 39
Convergent evolution Reappearance of ser-his-asp triad in unrelated settings
Subtilisin: externals very different from mammalian serine proteases; triad same
11/5/2009 Biochem: Specific Mechanisms P. 22 of 39
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.
11/5/2009 Biochem: Specific Mechanisms P. 23 of 39
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
11/5/2009 Biochem: Specific Mechanisms P. 24 of 39
Papain active site
Diagram courtesy Martin Harrison,Manchester University
11/5/2009 Biochem: Specific Mechanisms P. 25 of 39
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
11/5/2009 Biochem: Specific Mechanisms P. 26 of 39
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)
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The controversy
11/5/2009 Biochem: Specific Mechanisms P. 28 of 39
Triosephosphate isomerase(TIM) dihydroxyacetone phosphate glyceraldehyde-3-phosphate
Km=10µMkcat=4000s-1
kcat/Km=4*108M-1s-1
DHAP
Glyc-3-P
11/5/2009 Biochem: Specific Mechanisms P. 29 of 39
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)
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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
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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
11/5/2009 Biochem: Specific Mechanisms P. 32 of 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
11/5/2009 Biochem: Specific Mechanisms P. 33 of 39
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
11/5/2009 Biochem: Specific Mechanisms P. 34 of 39
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
11/5/2009 Biochem: Specific Mechanisms P. 35 of 39
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
11/5/2009 Biochem: Specific Mechanisms P. 36 of 39
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
11/5/2009 Biochem: Specific Mechanisms P. 37 of 39
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
11/5/2009 Biochem: Specific Mechanisms P. 38 of 39
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
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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