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Enzyme Catalysis: Understanding the Role of Enzymes in Biological Chemistry, Lecture notes of Biology

An overview of enzyme catalysis, including the functions and roles of enzymes as biological catalysts, the importance of cofactors and protein-ligand interactions, and the various types of catalysis. It also discusses the principles of catalysis, the energy profile of catalysis, and the mechanisms of enzyme catalysis.

What you will learn

  • What are the different types of catalysis and how do they differ?
  • What are enzymes and what role do they play in biological systems?
  • What are cofactors and what role do they play in enzyme catalysis?
  • How do enzymes accelerate chemical reactions?
  • How do protein-ligand interactions contribute to enzyme catalysis?

Typology: Lecture notes

2020/2021

Uploaded on 05/24/2021

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CHM 8304!
Enzyme Catalysis!1!
Enzyme Kinetics
Enzyme Catalysis
Outline: Enzyme catalysis
enzymes and non-bonding interactions (review)
catalysis (review - see section 9.2 of A&D)
general principles of catalysis
differential binding
types of catalysis
approximation
electrostatic
covalent
acid-base catalysis
strain and distortion
enzyme catalysis and energy diagrams
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Enzyme Kinetics

Enzyme Catalysis

Outline: Enzyme catalysis

  • enzymes and non-bonding interactions (review)
  • catalysis (review - see section 9.2 of A&D)
    • general principles of catalysis
    • differential binding
    • types of catalysis
      • approximation
      • electrostatic
      • covalent
      • acid-base catalysis
      • strain and distortion
  • enzyme catalysis and energy diagrams 2

Enzymes

  • proteins that play functional biological roles
  • responsible for the catalysis of nearly all chemical reactions that take place in living organisms - acceleration of reactions by factors of 10^6 to 10^17
  • biological catalysts that bind and catalyse the transformation of substrates
  • the three-dimensional structures of many enzymes have been solved (through X-ray crystallography) 3

Reminder: Amino acid structures

  • as proteins, enzymes are polymers of amino acids whose side chains interact with bound ligands (substrates) 4 R CO 2 H H 2 N H

Coenzymes

  • organic molecules, very often vitamins
    • e.g.: nicotinic acid gives NAD; pantothenic acid gives CoA
  • intermediates in the transport of functional groups
    • e.g. H (NAD), acyl (CoA), CO 2 (biotin), etc
  • also known as prosthetic groups 7

Protein-ligand interactions

  • covalent bonds
  • ionic bonds
  • ion-dipole and dipole-dipole interactions
  • hydrogen bonds
  • charge transfer complexes
  • hydrophobic interactions
  • van der Waals interactions 8

Covalent bond

  • the formation of a covalent bond can represent a stabilisation of 40 to 110 kcal/mol e.g.: N R H activé O R H 2 N 9 activated

Ionic bonds

  • Coulombic attraction between full positive and negative charges
    • ~5 kcal/mol of stabilisation NH 2 H OH (^) O 2 C

O 2 C

O

O

δ+^ N δ− 10

Charge transfer complex

  • special type of dipole-dipole interaction
  • involves π electrons, often in aromatic rings (Phe, Tyr, Trp, His)
    • stabilisation : < 3 kcal/mol 13

Hydrophobic interactions

  • stabilisation largely due to desolvatation (entropy increase)
    • stabilisation : ~0.5 kcal/mol CH 2 CH 2 H O H H O H H O H H O H H O H H surface + O (^) H hydrophobe H H^ O 6 (désolvatation) CH 2 CH 2 H O H H (^) O H H (^) O H H O H 14 hydrophobic surface (desolvation)

van der Waals interactions

  • special type of dipole-dipole interaction
    • movement of electrons in electron cloud of alkyl chains induces the formation of temporary dipoles
    • very important over short distances
    • stabilisation : ~0.5 kcal/mol (per interaction) 15

Outline: General principles of catalysis

  • see section 9.1 of A&D
    • principles of catalysis
    • differential bonding 16

Catalysis and free energy

  • catalysis accelerates a reaction by stabilising a TS relative to the ground state - free energy of activation, ΔG‡, decreases - rate constant, k , increases
  • catalysis does not affect the end point of an equilibrium, but only accelerates how quickly equilibrium is attained - free energy of the reaction, ΔG°, remains unchanged - equilibrium constant, K eq, remains unchanged 19

Energy profile of catalysis

20 Free energy Reaction coordinate Δ G‡uncat Δ G‡cat A Δ Gºrxn P unchanged uncatalysed A • cat P • cat catalysed

Transition state binding

  • interaction between a catalyst and reactant or activated complex can stabilise one or the other
  • if the activated complex is bound more strongly than the substrate, the activation barrier will be decreased
  • HOWEVER, the activated complex is not a molecule – so the catalysts must first of all interact with the substrate, and then release the product at the end of the reaction : A + cat A•cat P•cat (^) P + cat 21

Differential binding

  • consider 4 scenarios : Energy Rxn. coord. Δ G‡non-cat Energy Rxn. coord. Δ G‡non-cat Energy Rxn. coord. Δ G‡non-cat Energy Rxn. coord. Δ G‡non-cat Δ G‡cat binding binding Δ G‡cat binding Δ G‡cat binding binding Δ G‡cat binding 22

Catalysis by approximation

  • the catalyst brings together the reactants, increasing their effective concentrations , and orients them with respect to the reactive groups Jencks :
  • the loss of entropy associated with the restriction of rotation and translation of substrate must be compensated by the intrinsic energy of binding (favourable non-bonding interactions) Bruice / Kirby :
  • the magnitude of this effect is given by the effective concentration , determined by comparison if the rate constants of the bimolecular and intramolecular reactions 25

Intramolecular approximation

  • an intramolecular reaction implies a smaller decrease in entropy (and therefore a decrease in the free energy of activation) Coordonnée de réaction Énergie libre ΔG réactifs A + B A B ΔG = ΔH - TΔS intermolecular intramolecular Reaction co-ordinate reactants Free energy (^) note trade-off between enthalpy (in bond formation) and entropy 26

Example of catalysis by approximation

  • the catalyst brings together the reactants, increasing their effective concentrations , and orients them with respect to the reactive groups k (^1) obs k 2 obs = 5000 M = la^ concentration effective O 2 N O O CH 3
  • O 2 N^ O- O CH 3 (H 3 C) 3 +N

(H 3 C) 2 NCH 3 k obs 2 = 4,3 M-1s- O 2 N O O CH 2 CH 2 (H 3 C) 2 NCH 2 O 2 N (^) O- O CH 2 (H 3 C) 2 +N

k obs 1 = 21 500 s- 27 effective concentration , or effective molarity (EM)

Example: Ester hydrolysis

Réaction Constante de vitesse relative ( k rel) Concentration effective (M) O OAr

CH 3 CO 2 - 1 M-^1 s-^1 - O OAr O- O 220 s-^1 220 M O OAr O- O 5.1 × 104 s-^1 5.1 × 104 M O OAr O- O 2.3 × 106 s-^1 2.3 × 106 M O O OAr O O-^ 1.2^ ×^107 s-^1 1.2^ ×^107 M decreasing entropy of rotation and translation parent reaction

Reaction k rel

Effective concentration (M) 28

Electrostatic catalysis

  • e.g.: oxyanion hole of subtilisin: J. Mol. Biol. 1991 , 221 , 309-325. (^) 31

Electrostatic catalysis

  • can be very important :
    • consider the triple mutant of subtilisin where each residue of its catalytic triad is replaced (S221A-H64A-D32A) - catalyses proteolysis 10^6 -fold less than the native enzyme - BUT the reaction with the mutant is still 10^3 -fold faster than the uncatalysed reaction!! - an important part of catalysis is due to the electrostatic environment 32

Metal catalysis

  • electrostatic charges developed at the TS can also be stabilised by metal ions
  • coordination of a ligand by a metal (as a Lewis acid) can also lead to polarisation of a ligand - e.g. p K a of metal-bound H 2 O is 7.2, making it easier to deprotonate, therby generating – OH as a nucleophile - for example, zinc-bound water in carbonic anhydrase, a highly efficient metalloenzyme as well as certain enzyme models 33

Covalent catalysis

  • catalyst forms a covalent intermediate that reacts faster than uncatalysed reaction : A + B P vs intermédiare A + B + C AC^ + B^ P + C more reactive intermediate 34

Nucleophilic catalysis

  • catalyst attacks substrate to form intermediate that is even more susceptible to nucleophilic attack, by a second reactant - e.g. reaction of acid chlorides with alcohols, catalysed by addition of a tertiary amine: R Cl O (^) R'OH R O O (lente)^ R' R N O Et Et Et R'OH (rapide) NEt (^3) NEt 3 Energy Rxn. coord. Δ G‡uncat Δ G‡cat 37

Acid-base catalysis

  • the catalyst (namely an acid or a base) accelerates the reaction through protonation or deprotonation 38

Specific acid-base catalysis

  • catalysis by H+^ or – OH, controlled only by pH, where a fast equilibrium precedes the rls : - e.g.: 39 O O O O H O O H H^ O^ H^ O O H H^ O^ H OH O H HO

OH O

  • H+ fast slow H 2 O H+

Rate laws of specific acid-base catalysis

  • when a substrate must be protonated before its reaction in the rls, this appears as a pH dependence in the rate law: - e.g.: v = k [R]×[H+]/ K a,RH
  • when a substrate must be deprotonated before its reaction in the rls, this appears as a pH dependence in the rate law: - e.g.: v = k [RH]× K a,RH /[H+] 40