LSM1101 LECTURE 1

ENZYMES

Dr Deng Lih Wen

Dept of Biochemistry

Overview

Lecture 1: EnzymesLecture 2: Enzyme KineticsLecture 3: Enzyme RegulationLecture 4: Myoglobin/Hemoglobin

Practical

Tutorial

* Tutorial 3 (Group 1+2): 6pm to 7pm on Sep 18th

Recommended books

Fundamentals of Biochemistry 2nd Ed. by D Voet, J. G. Voet & C. W. Pratt

Biochemistry 3rd Ed. by R.H. Garrett & C.M. Grisham

Lehninger Principles of Biochemistry 4th Ed, David L. Nelson & Michael M. Cox

Lippincott’s Illustrated Reviews in Biochemistry 3rd Ed. by P. C. Champe, R. A. Harvey & D. R. Ferrier

Lecture outline

General Properties of EnzymesClassificationSubstrate specificityCofactors

Activation Energy and the Transition State Diagram

Catalytic MechanismAcid-BaseCovalent Metal Ion

What is Enzyme?An enzyme is a protein catalyst that increase the velocity of a chemical reaction and is not consumed during the reaction it catalyzes. Exception: Ribozyme - RNA acts like enzymes, usually catalyzing the cleavage and synthesis of phosphodiester bonds.

Active site – region that binds substrate and converts it into product3D conformation usually forming a cleftBinding of substrate by multiple weak forces

Enzyme Classification

Systematic name by International Union of Biochemistry and Molecular Biology (IUBMB) http://expasy.org/enzyme

2 H2O2 2 H2O + O2

Traditionally, enzymes are named by adding the suffix –ase to the name of the substrate or to a description of the action performed. Eg. Urease, Alcohol dehydrogenase

Catalase???

Enzyme commission class, subclass, sub-subclass, individual entry

EC 3. 4. 17. 1

Six Major Classes by IUBMB

Enzyme Properties

Higher Reaction RatesMilder Reaction ConditionsGreater SpecificityCapacity for Regulation

NO other phospho-glucose (e.g. glucose-1-phosphate, or glucose-3-phosphate) is produced during the reaction

Specificity ensures that the final product is not contaminated with by-products.

Overall yield over 10 stepsYield per step

50% ? %

90% 34.9%

97% ? %

Enzymes produce products in very high yields - often much greater than 95%

Models of Active Site Interaction

1. Lock-and-Key Model(Emil Fisher, 1894)

2. Induced-Fit Model (Daniel Koshland, 1958)The binding of the substrate induces a conformational change in the enzyme that results in a complementary fit once the substrate is bound. The binding site has a different 3-D shape before the substrate is bound

http://scholar.hw.ac.uk/site/biology/activity6.asp

CofactorsSome enzymes are associated with non-protein componentswhich are required for enzyme activity.

Inorganic metal ions:

Organic molecules (also called coenzymes), often derivatives of vitamins

Apoenzyme + cofactor Holoenzymes

Prosthetic group: a subset of cofactors. may be organic or inorganic or both

bound tightly to proteins and may even be attached through a covalent bond.

(inactive) (active)

Cofactors: Metal ions

Principles of Biochemistry 4th Ed

Cofactors: Coenzymes

Ethanol Acetaldehyde

Alcohol dehydrogenase

Many Vitamins Are Coenzyme Precursors

niacytin (in corn)

beriberihttp://mywebpages.comcas.net/swaneyj/Netrition/B_Vitamins/p1.htm

Vitamin B3

Effect of pH on Enzyme Activity

Usually there is an optimum pH for maximum activity

pH can affects ionization of amino acid side groups Change in substrate-active site interactionLeading to change in enzyme activity

Effect of Temperature on Enzyme Activity

Lecture outline

General Properties of EnzymesClassificationSubstrate specificityCofactors

Activation Energy and the Transition State Diagram

Catalytic MechanismAcid-BaseCovalent Metal Ion

Reaction coordinate diagram

A + B → X ‡ → P + Q

∆G‡ & ∆Greaction

Gibbs Free Energy: Takes into account both enthalpy and entropy. (G = H – TS)Transition state: the point of highest free energy; very unstable; cannot be isolated∆G‡ : the free energy of activation, the difference in free energy between the reactants andthe transition state∆Greaction : free energy of the products minus the free energy ofreactants.

∆Greaction & Reaction Equilibrium

Reactant Product

K’eq = [ Product]

[ Reactant]

∆Greaction= - RT ln K’eq

R: gas constant 8.315J/mol·KT: absolute temperature, 298K (25ºC)

•∆G reaction < 0: the equilibrium favours products.

•A large negative value of ∆G reactionindicates that the reaction has a strong tendency to occur.

K’eq ∆Greaction (kJ/mol)

Glucose + 6 O2 –> 6 CO2 + 6 H2O∆Greaction= - 2880KJ/mole

Oxidation of glucose is strongly exergonic, but it doesn’t occur under normal conditions in air with an unlimited supply of oxygen.

Why???

Can the Direction of Equilibrium be affected by Enzymes?

What Does the Enzyme Change?

Enzymes Reduces Activation Energy

In the presence of enzyme – hill is lower

cat

uncat

Figure 13.1Reaction profile showing large ΔG‡ for glucose oxidation, free energy change of -2,870 kJ/mol; catalysts lower ΔG‡, thereby accelerating rate.

Enzymes Reduce ∆G‡ but have NO effects on ∆Greaction

Decrease the free energy barrier (activation energy, ∆G‡) to allow the reaction to approach equilibrium more quickly Accelerates both the forward and reverse reactionsCAN NOT alter the free energy change of the reaction (∆Greaction)

∆Greaction < 0, the equilibrium favours products.∆Greaction > 0, the equilibrium favours reactants.

Rethink of Lock-and-Key model

Fig 4.43, Textbook of Biochemistry with Clinical correlations, 4th Ed

Is an Enzyme completely complementary to its substrate a very good enzyme?

An Enzyme Completely Complementary to Its substrate Would be a Very Poor Enzyme

Fig 6-7, Principles of Biochemistry 4th Ed

An Enzyme must be complementary to the reaction transition state. The optimal interactions (through weak bonding) between substrate and enzyme can occur only in the transition state (Haldane 1930; Linus Pauling 1946.)

Lecture outline

General Properties of EnzymesClassificationSubstrate specificityCofactors

Activation Energy and the Transition State Diagram

Catalytic MechanismAcid-BaseCovalent Metal Ion

Acid-Base Catalysis

uncatalyzed

Acid catalyzed

Base catalyzed

Enzyme can make some atoms or functional group in their substrate more reactive by adding a proton or removing a proton from them.

Amino acids in general acid-base catalysis

Lehninger Fig 6.9

RNase A: Hydrolyze RNA

+H

pK (COOH) 1.8

pK (NH3+) 9.3

pK (imidazole group) 6.0

1. His 12, acting as a general baseHis 119, acting as a general acid

2. After the leaving group departs, water enters the active site, reverse of stage 1.His 12, acting as a general acidHis 119, acting as a general base

2

Catalytic Mechanisms

Acid-base catalysis

Covalent catalysis

Metal ion catalysis

Covalent catalysis

Involves the transient formation of a catalyst-substrate covalent bond

Nucleophilic attack: the enzyme is a nucleophile and the substrate is the electrophilelead to covalent bond formation

Biologically Important Nucleophiles and Electrophiles

Nucleophilic catalysis resembles base catalysis except that instead of abstracting a proton from the substrate, the catalyst attacks the substrate to form a covalent bond.

negative charged orElectron rich

contain an electron-deficient atom (shown in red)

Covalent Catalysis (Nucleophilic Catalysis)

Transient formation of a catalyst-substrate covalent bondThe more stable the covalent bond formed, the less easily it candecompose in the final step of a reaction

Uncat

1. The nucleophilicreaction between the catalyst and the substrate to form a covalent bond

2. The withdraw of electrons from the reaction center by the electrophilic catalyst.

3. Elimination of the catalyst, reverse of stage 1

Catalytic Mechanisms

Acid-base catalysis

Covalent catalysis

Metal ion catalysis

Metal Ion Catalysis

Nearly one-third of known enzymes require the presence of metal ions for catalytic activity.Four major ways

Binding to substrates to orient them properly for reactionElectrostatically stabilizing or shielding negative chargesPolarization of substrates Mediating oxidation-reduction reactions

Orientation of substrate

CH3

Stabilize or shield negative charges

Role of Mg++: Shield the negative charges of the phosphate groups (these negative charges tend to repel the electron pairs of attacking nucleophiles)

Mg++..

•His64 is too far away from the zinc-bound water to directly abstract its proton

•A hydrogen bond network is formed by linking two intervening water molecules.

Binding to zinc leads to the polarization of water which is facilitated through a proton shuttle.

Promote nucleophilic catalysis via water ionization

CO2 + H2O H+ + HCO3-

Carbonic anhydrase

Carbonic AnhydraseCO2 + H2O H+ + HCO3-

1. His64 de-protonate H2O to give OH- which is held by the Zn2+

2. Zn2+ -bound OH-

nucleophilically attacks the nearby enzymatically bound CO2, converting it to HCO3

-

3. The catalytic site is then regenerated by the binding and ionization of another H2O at the Zn2+