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Dr Deng Lih Wen Dept of Biochemistry

Lecture 1: Enzymes Lecture 2: Enzyme Kinetics Lecture 3: Enzyme Regulation Lecture 4: Myoglobin/Hemoglobin Practical Tutorial
* Tutorial 3 (Group 1+2): 6pm to 7pm on Sep 18th

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

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 Enzymes Classification Substrate specificity Cofactors Activation Energy and the Transition State Diagram Catalytic Mechanism Acid-Base Covalent Metal Ion

RNA acts like enzymes. usually catalyzing the cleavage and synthesis of phosphodiester bonds.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 . Active site – region that binds substrate and converts it into product 3D conformation usually forming a cleft Binding of substrate by multiple weak forces .

individual entry . Alcohol dehydrogenase Catalase??? 2 H2O2 2 H2O + O2 Systematic name by International Union of Biochemistry and Molecular Biology (IUBMB) http://expasy. 1 Enzyme commission class. 17. sub-subclass. enzymes are named by adding the suffix –ase to the name of the substrate or to a description of the action performed. 4. Eg.Enzyme Classification Traditionally. subclass. EC 3.

Six Major Classes by IUBMB .

Enzyme Properties Higher Reaction Rates Milder Reaction Conditions Greater Specificity Capacity for Regulation NO other phospho-glucose (e. glucose-1-phosphate. or glucose-3-phosphate) is produced during the reaction .g.

Specificity ensures that the final product is not contaminated with by-products.9% ? % Enzymes produce products in very high yields .often much greater than 95% . Yield per step 50% 90% 97% Overall yield over 10 steps ? % 34.

uk/site/biology/ Lock-and-Key Model (Emil Fisher. The binding site has a different 3-D shape before the substrate is bound http://scholar.asp . 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.Models of Active Site Interaction 1.hw.

may be organic or inorganic or both bound tightly to proteins and may even be attached through a covalent bond. Inorganic metal ions: Organic molecules (also called coenzymes). often derivatives of vitamins Apoenzyme + cofactor (inactive) Holoenzymes (active) Prosthetic group: a subset of cofactors. .Cofactors Some enzymes are associated with non-protein components which are required for enzyme activity.

Cofactors: Metal ions Principles of Biochemistry 4th Ed .

Cofactors: Coenzymes Alcohol dehydrogenase Ethanol Acetaldehyde .

net/ swaneyj/Netrition/B_Vitamins/p1.Many Vitamins Are Coenzyme Precursors Vitamin B3 beriberi niacytin (in corn) http://mywebpages.comcas.htm .

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 interaction Leading to change in enzyme activity .

Effect of Temperature on Enzyme Activity .

Lecture outline General Properties of Enzymes Classification Substrate specificity Cofactors Activation Energy and the Transition State Diagram Catalytic Mechanism Acid-Base Covalent Metal Ion .

very unstable.∆G & ∆Greaction A + B → X ‡ ‡ → P+Q Reaction coordinate diagram Gibbs Free Energy: Takes into account both enthalpy and entropy. the difference in free energy between the reactants and the transition state ∆Greaction : free energy of the products minus the free energy of reactants. . (G = H – TS) Transition state: the point of highest free energy. cannot be isolated ∆G‡ : the free energy of activation.

•A large negative value of ∆G reaction indicates that the reaction has a strong tendency to occur. .∆Greaction & Reaction Equilibrium Reactant Product [ Product] [ Reactant] ∆Greaction= .315J/mol·K T: absolute temperature.RT ln K’eq R: gas constant 8. 298K (25ºC) K’eq = K’eq ∆Greaction (kJ/mol) •∆G reaction < 0: the equilibrium favours products.

Why??? . but it doesn’t occur under normal conditions in air with an unlimited supply of oxygen.Glucose + 6 O2 –> 6 CO2 + 6 H2O ∆Greaction= .2880KJ/mole Oxidation of glucose is strongly exergonic.

Can the Direction of Equilibrium be affected by Enzymes? What Does the Enzyme Change? .

Enzymes Reduces Activation Energy uncat cat In the presence of enzyme – hill is lower .

free energy change of -2.1 Reaction profile showing large ΔG‡ for glucose oxidation. . thereby accelerating rate. catalysts lower ΔG‡.870 kJ/mol.Figure 13.

∆Greaction > 0.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 reactions CAN NOT alter the free energy change of the reaction (∆Greaction) ∆Greaction < 0. the equilibrium favours products. the equilibrium favours reactants. .

43.Rethink of Lock-and-Key model Is an Enzyme completely complementary to its substrate a very good enzyme? Fig 4. 4th Ed . Textbook of Biochemistry with Clinical correlations.

An Enzyme Completely Complementary to Its substrate Would be a Very Poor Enzyme Fig 6-7. The optimal interactions (through weak bonding) between substrate and enzyme can occur only in the transition state (Haldane 1930. Principles of Biochemistry 4th Ed An Enzyme must be complementary to the reaction transition state.) . Linus Pauling 1946.

Lecture outline General Properties of Enzymes Classification Substrate specificity Cofactors Activation Energy and the Transition State Diagram Catalytic Mechanism Acid-Base Covalent Metal Ion .

Acid-Base Catalysis Enzyme can make some atoms or functional group in their substrate more reactive by adding a proton or removing a proton from them. uncatalyzed Acid catalyzed Base catalyzed .

Amino acids in general acidbase catalysis Lehninger Fig 6.9 .

3 pK (imidazole group) 6.RNase A: Hydrolyze RNA pK (COOH) 1.0 +H .8 pK (NH3+) 9.

acting as a general acid 2. acting as a general base . After the leaving group departs. reverse of stage 1. His 12.2 1. acting as a general acid His 119. water enters the active site. His 12. acting as a general base His 119.

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 electrophile lead to covalent bond formation .

Biologically Important Nucleophiles and Electrophiles negative charged or Electron rich contain an electron-deficient atom (shown in red) 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.

Covalent Catalysis (Nucleophilic Catalysis) Uncat 1. reverse of stage 1 Transient formation of a catalyst-substrate covalent bond The more stable the covalent bond formed. the less easily it can decompose in the final step of a reaction . 3. The nucleophilic reaction between the catalyst and the substrate to form a covalent bond 2. Elimination of the catalyst. The withdraw of electrons from the reaction center by the electrophilic catalyst.

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 reaction Electrostatically stabilizing or shielding negative charges Polarization of substrates Mediating oxidation-reduction reactions .

Orientation of substrate CH3 .

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

.Promote nucleophilic catalysis via water ionization Carbonic anhydrase CO2 + H2O H+ + HCO3- •His64 is too far away from the zincbound 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.

which is held by the Zn2+ 2. Zn2+ -bound OHnucleophilically attacks the nearby enzymatically bound CO2. The catalytic site is then regenerated by the binding and ionization of another H2O at the Zn2+ . converting it to HCO33. His64 de-protonate H2O to give OH.Carbonic Anhydrase CO2 + H2O H+ + HCO31.