1. Enzymes: mechanisms & regulation
Andy Howard Introductory Biochemistry
5 November 2013
Enzyme Mechanisms
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2. More about mechanisms
Many enzymatic mechanisms involve either covalent catalysis or acid-base interactions
We’ll give some examples of several mechanistic approaches
Then we’ll talk about enzyme activity is regulated
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3. Mechanism Topics Other mechanisms Redox reactions Induced fit
Cysteinyl proteases
Lysozyme
TIM
Regulation: thermodynamics
Enzyme availability
Transcription
Degradation
Compartmentation
Allostery
Mechanisms
Kinetics
PTM
Redox reactions
Induced fit
Ionic intermediates
Active-site amino acids
Serine proteases
Reaction
How they illustrate what we’ve learned
Specificity
Evolution
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4. Oxidation-Reduction Reactions
Commonplace in biochemistry: EC 1
Oxidation is a loss of electrons
Reduction is the gain of electrons
In practice, often:
oxidation is decrease in # of C-H bonds;
reduction is increase in # of C-H bonds
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5. Redox, continued
Intermediate electron acceptors and donors are organic moieties or metals
Ultimate electron acceptor in aerobic organisms is usually dioxygen (O2)
Anaerobic organisms usually employ other electron acceptors
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6. Biological redox reactions
Generally 2-electron transformations
Often involve alcohols, aldehydes, ketones, carboxylic acids, C=C bonds:
R1R2CH-OH + X  R1R2C=O + XH2
R1HC=O + X + OH- R1COO- + XH2
X is usually NAD, NADP, FAD, FMN
A few biological redox systems involve metal ions or Fe-S complexes
Usually reduced compounds are higher-energy than the corresponding oxidized compounds
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7. One-electron redox reactions
FMN
One-electron redox reactions
FMN, FAD, some metal ions can be oxidized or reduced one electron at a time
With organic cofactors this generally leaves a free radical in each of two places
Subsequent reactions get us back to an even number of electrons
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8. Covalent catalysis
Reactive side-chain can be a nucleophile or an electrophile; nucleophile is more common
A—X + E  X—E + A
X—E + B  B—X + E
Example: sucrose phosphorylase
Net reaction: Sucrose + Pi  Glucose 1-P + fructose
Fructose=A, Glucose=X, Phosphate=B
Bifidobacterium sucrose phosphorylase
EC kDa dimer
PDB 1R7A, 1.8Å
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9. Example: hexokinase Glucose + ATP  Glucose-6-P + ADP
Human brain Hexokinase I EC
104 kDa monomer
PDB 1CZA, 1.9Å
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
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10. Hexokinase structure Diagram courtesy E. Marcotte, UT Austin
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11. 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
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12. Reactive sidechains in a.a.’s
Group
Functions
Asp
—COO- -1
Cation binding, H+ transfer
Glu
Same as above
His
Imidazole ~0
Proton transfer
Cys
—CH2SH
Covalent binding of acyl gps
Tyr
Phenol
H-bonding to ligands
Lys
NH3+ +1
Anion binding, H+ transfer
Arg
guanidinium
Anion binding
Ser
—CH2OH
See cys
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13. Generalizations about active-site amino acids
Typical enzyme has 2-6 key catalytic residues
His, asp, arg, glu, lys account for 64%
Remember:
pKa values in proteins sometimes different from those of isolated amino acids
Frequency overall  Frequency in catalysis
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14. Rates often depend on pH
If an amino acid that is necessary to the mechanism changes protonation state at a particular pH, then the reaction may be allowed or disallowed depending on pH
Two ionizable residues means there may be a narrow pH optimum for catalysis
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15. Papain as an example
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16. iClicker quiz, question 1
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
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17. iClicker Quiz question 2
2. Triosephosphate isomerase (TIM) interconverts dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate. 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
(e) None of them would bind at all.
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18. 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
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19. 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 amino acids at position 1, 2, -1, . . .
Others are more promiscuous O CH NH C NH C NH R1 CH O
R-1
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20. Mechanism
Active-site serine —OH … Without neighboring amino acids, it’s fairly unreactive
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.
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21. 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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22. Diagram of first three steps
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23. Diagram of last four steps
Diagrams courtesy University of Virginia
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24. 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
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25. 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.
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26. Oxyanion hole cartoon
Cartoon courtesy Henry Jakubowski, College of St.Benedict / St.John’s University
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27. 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
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28. 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
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29. 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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30. 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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31. 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
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32. iClicker quiz, question 3
3. 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
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33. iClicker question 4
4. Which of these enzymes would you predict to be the most similar to human pancreatic elastase?
(a) human pancreatic chymotrypsin
(b) porcine pancreatic elastase
(c) subtilisin from Bacillus subtilis
(d) none of these would be very similar to human pancreatic elastase
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34. Convergent evolution
Reappearance of ser-his-asp triad in unrelated settings
Subtilisin: externals very different from mammalian serine proteases; triad same
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35. 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.
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36. Cysteinyl proteases Ancestrally related to serine proteases?
Cathepsins, caspases, papain
Contrasts:
Cys —SH is more basic than ser —OH
Residue is less hydrophilic
S- is a weaker nucleophile than O-
Diagram courtesy of Mariusz Jaskolski, U. Poznan
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37. Papain active site
Diagram courtesy Martin Harrison, Manchester University
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38. 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
HEWL PDB 2vb1 0.65Å 15 kDa
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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
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40. The controversy
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41. Triosephosphate isomerase (TIM)
dihydroxyacetone phosphate 
glyceraldehyde-3-phosphate
Glyc-3-P
DHAP
Km=10µM
kcat=4000s-1
kcat/Km=4*108M-1s-1
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42. 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 ( CHY431/Enzyme3.html)
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43. 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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44. 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
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45. 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
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46. 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
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47. 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
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48. 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
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49. 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
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50. 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
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51. 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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52. 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
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53. 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 sequence that resembles target sequence; this binds, inactivates the kinase’s catalytic subunit
When regulatory subunits bind cAMP, it releases from catalytic subunit so it can perform
Mouse cAMP-dependent protein kinase
EC kDa dimer of dimers PDB 3TNP, 2.3Å
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54. 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
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55. 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
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56. MWC model for allostery
Emphasizes symmetry and symmetry-breaking in seeing how subunit interactions give rise to allostery
Can only explain positive cooperativity
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57. Koshland (KNF) model
Emphasizes conformational changes from one state to another, induced by binding of effector
Ligand binding and conformational transitions are distinct steps
… so this is a sequential model for allosteric transitions
Allows for negative cooperativity as well as positive cooperativity
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58. Oligomerization and allostery
Often the RT transition is influenced by enzyme oligomerization
Tryptophan synthase: dimerization shifts equilibrium toward R
Amino acid kinases: hinge motions of monomers engage cooperativity
Thermatoga N-acetylglutamate kinase: 194 kDa hexamer, trimer shown EC ; PDB 2BTY, 2.75¸Å
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59. Heterotropic effectors
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60. Post-translational modification
We’ve already looked at phosphorylation
Proteolytic cleavage of the enzyme to activate it is another common PTM mode
Some proteases cleave themselves (auto-catalysis); in other cases there’s an external protease involved
Blood-clotting cascade involves a series of catalytic activations
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61. Zymogens
As mentioned earlier, this is a term for an inactive form of a protein produced at the ribosome
Proteolytic post-translational processing required for the zymogen to be converted to its active form
Cleavage may happen intracellularly, during secretion, or extracellularly
Bacillus subtilisin + prosequence
35 kDa heterodimer EC
PDB 3CNQ, 1.71Å
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62. Blood clotting Seven serine proteases in cascade
Final one (thrombin) converts fibrinogen to fibrin, which can aggregate to form an insoluble mat to prevent leakage
Two different pathways:
Intrinsic: blood sees injury directly
Extrinsic: injured tissues release factors that stimulate process
Come together at factor X
Human thrombin EC
36kDa monomer PDB 3RM2, 1.23Å
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63. Cascade Human Factor Xa EC 3.4.21.6 34kDa monomer PDB 2JKH, 1.25Å
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64. Protein-protein interactions
One major change in biochemistry in the last 25 years is the increasing emphasis on protein-protein interactions in understanding biological activities
Many proteins depend on exogenous partners for modulating their activity up or down
Example: cholera toxin’s enzymatic component depends on interaction with human protein ARF6
Vibrio cholerae toxin A1 subunit + human ARF6: 37 kDa heterodimer PDB 2A5D, 1.8Å
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