Enzymes Flashcards
Enzyme-substrate complementarity
Substrate and residues in active site as close together as possible for maximum binding energy (snug fit)
Greater complementarity + greater binding energy
Factors involved in binding substrate
H bonds
Electrostatic
Hydrophobic
VDW
What is Kcat
Turnover number
What is Kcat/Km
Indicated whether an enzyme-catalysed reaction is diffusion controlled
I.e. chemical+release steps are so fast that enzyme is just waiting to encounter substrate
10^8 to 10^9 M-1S-1
Transition state theory
Transition state represents the most unstable species in a reaction pathway where chemical bonds are being broken and made and is located at the peak of the reaction profile
Intermediates have fully formed bonds and occupy troughs in the profile
Kcat/Kuncat = e^change in binding energy/RT
Active site of an enzyme has evolved to bind TS more tightly than the substrate
Gives maximum binding energy
General acid catalysis
Process whose rate is dependent on the concentration of all acids present in the reaction not just {protons] of aqueous reactions, e.g. a side chains
General base catalysis
Process whose rate is dependent on the concentration of all bases present in the reaction, not just [OH-] if aqueous reaction
Specific acid / base catalysis
Reactions whose reactions are just dependent on [H+] or [OH-]
Concerted general acid-base catalysed reaction
Both processes occur simultaneously, only one TS, proton delivered and accepted at same time
PH dependency of acid-base catalysed reactions
Reversible reaction where aa side chains are deprotonated
Above pKa= deprotonated
Must be in correct protonation state for enzyme to be active
Effect of environment on pKa values
PKa values can be shifted by micro environment
Polarity + presence of charges
Hydrophobic region = uncharged species favoured = increase pKa
+ve charge nearby = -ve charge stabilised = decreased pKa
-ve charge nearby = -ve charge destabilised = increased PKa
Covalent catalysis
Formation of covalently bound enzyme intermediate
Form from reaction of the substrate with an aa side chain or a coenzyme
Aa side chains can act as nucleophiles but only reactive when deprotonated
Important in group transfer reactions - 2 substrate reactions that usually involve transfer of one group from one substrate to another
Important in making substrate more reactive
E.g. formation of schifi bases to activate carbonyl groups
chemical trapping of enzyme intermediates
React enzyme with NaBH4, source of hydride ions
Forms amine, non-reactive , prevents further catalysis
Enzyme inactivated as modified form of substrate is permanently attached to Lys side chain
Electrostatic catalysis
Presence of charges and oriented dipoles within the active site can stabilise TS by solvation
TS accumulates charge, surround TS with opposite charge to get favourable charge-charge interactions to get binding energy
Coulombic interactions are more effective in non-polar environments as they have lower dielectric constant
Metal ion catalysis
Metalloenzymes containing tightly bound ions e.g Fe2+ or Mg2+
Metal-activated enzymes loosely bind ions from solution e.g. Na+ , K+
What are metal ions involved in
Electrostatic stabilisation and charge screening
Highly effective because of high charge that is pH independent
E.g. Mg2+-ATP complex in kinases
Redox reactions
Binding and orientation of substrates
Act as Lewis acids and can form dative or covalent coordinate bonds
Promote nucleophilic catalysis by activating water bound molecules
Catalysis through orientation and proximity effects
Rate enhancement obtained by taking 2 reactants out of solution and placing them next to each other in the enzyme, raising the local concentration of each reactant
Binds substrate in precise geometry so orbitals have correct orientation
Entropy changes in bimolecular reactions
A + B —> A-B has large -ve delta s
A + B —> AB in enzyme —> A-B smaller -ve S so more favourable
Enzyme can tether reactants together to increase local conc
Serine protease catalytic triad
Asp-102
His-57
Ser-195
Serine protease mechanism
1)Nucleophilic attack of serine to form first tetrahedral intermediate
His-57 general base
Asp-102 electrostatic effect
2) decomposition of first tetrahedral intermediate to give Acyl-enzyme intermediate
His-57 general acid to form amine leaving group
3) His-57 acting as general base promotes nucleophilic attack by water on the Acyl-enzyme to form second tetrahedral intermediate
4) decomposition of intermediate to give resting enzyme and carboxylic acid
Protonated his-57 acts as a general acid
Serine protease oxyanion hole
Acts to stabilise transition state
In michaelis complex, trigonal carbon of scissile peptide bond is conformationally constrained from binding into oxyanion hole
In tetrahedral intermediate, the oxyanion species enters the hole and makes favourable H bonds to the backbone NH groups of Gly193 and ser 195
An additional H bond to gly193 is also made
Favourable binding interactions reduce energy of activation
Carbonyl oxygen shifts into cavity
Charged group stabilised by residues in enzyme
Only occurs in TS
Specificity of serine proteases
Determined by nature of S1 pocket and p1 residues of substrate
Chymotrypsin cleaves bulky P1, Phe, trp, Tyr
Trypsin has asp in pocket so cleaves +ve P1, Arg, Lys
Elastase has 2 valines in pocket s cleaves small, neutral P1, Ala, gly, ser, val
Divergent vs. Convergent evolution
Divergent is after a gene duplication event
Only need one copy of the gene so now second can mutate without causing harm
Produced trypsin, chymotrypsin, elastase
Convergent evolution produces same triad but with completely separate route
Kinetics of chymotrypsin
Shown with pre-steady state kinetics
Fast initial burst of product to generate covalently attached Acyl-enzyme intermediate
Slower regeneration of enzyme is RDS of catalytic cycle and limits catalysis in subsequent turnovers
Cysteine proteases
Similar to serine
Use thiol group which has lower pKa than ser
No aspartate as it doesnt need a base to pull off the proton
Aspartyl proteases
Water attacks directly
Aspartate in conjugate base form to pull off a proton
Second asp in protonated form ti H bond with oxyanion species to stabilise it
2 asp residues must have different pKa values
Metallo proteases
Similar to carbonic anhydrase
Tightly bound metal ion can bind water
Appropriately positions H2O to attack
Base close by to pull off proton
Electrostatic interaction between Zn2+ and oxyanion
