Unit 1: Introduction to Enzymes Introduction & Definition of Enzymes, Coenzymes, Cofactors, Apoenzymes, Holoenzyme, Abzymes, Synzyme, Ribozyme, Extremozyme, Historical Development in Enzymology, Characteristics of enzymes, Enzyme Vs Chemical Catalysts, Enzyme mechanism & Activation Energy, Enzyme Specificity, Fischer and Koshland Models Role of B complex Vitamins in Enzyme catalyzed reactions Multienzyme Complex: Properties, Examples with Reactions (PDH Complex, Fatty Acyl Synthase Complex, Tryptophan Synthase), Physiological Importance, Advantages of MEC Zymogens, Properties with examples and Activation of Chymotrypsinogen

What are Enzymes? Lecture 2

To understand the nature of enzymes Objective To understand the nature of enzymes

all the activities we characterize as “life” The living cell: site of tremendous biochemical activity called METABOLISM Activities: Build up new tissues, Replacement of old tissues, Conversion food to energy, Disposal of waste materials, Reproduction all the activities we characterize as “life” Activities require catalysts Catalyst is defined as the acceleration of a chemical reaction by some substance which itself undergoes no permanent chemical change

The catalysts of biochemical reactions are enzymes Enzymes are responsible for bringing about almost all the chemical reactions in living organisms Proteins have a vast capacity to bind to a wide range of molecules Using various intermolecular forces, enzymes bring substrates together in an optimal orientation for reaction Chemical reactions need an initial input of energy = THE ACTIVATION ENERGY They catalyse reaction by stabilizing transition state (highest energy species in reaction pathways) By stabilizing transition state, an enzyme determines which one of several potential chemical reaction actually takes place Without enzymes, these reactions take place at the rate far too slow for the pace of metabolism

Rate enhancement by selected enzymes Nonenzymatic half life Uncatalysed rate (Kun/s) Catalysed rate (Kcat/s) Rate enhancement (kcat/se kun/se) Orotidine Monophosphate decarboxylase 78,000,000 years 2.8 x 10-16 39 1.4 x 1017 AMP Nucleosidase 69,000 years 1.0 x 10-11 60 6.0 x 1012 Triose Phosphate Isomerase 1.9 years 4.3 x 10-6 4300 1.0 x 109 Carbonic anhydrase 5 seconds 1.3 x 10-1 1 x 106 7.7 x 106 A. Radzicka and R Wolenden, Science 267: 90-93, 1995

3 Characteristics of enzymes The most efficient catalysts known Specificity of action Many enzymes are regulated The individuality of a living cell is due in large part to the unique set of enzymes that it is genetically programmed to produce. If even one is missing or defective, the result can be disastrous Robert Bohinsky, Fundamentals of Biochemistry, 5th ed, 1987

Nature of Enzymes 1. Nearly all known Enzymes are proteins They have a globular shape A complex 3-D structure Human pancreatic amylase © Dr. Anjuman Begum

German scientist Richard Willstatter (Nobel Prize in 1915) reported in 1920 that he could not detect protein in purified enzyme preparation. This is corrected by Sumner

Evidence providing the protein nature of enzymes In their elementary composition, enzymes show the usual proportion of C,H,N and S as found in proteins. Enzymes are subjected to the action of those enzymes which are specifically meant for the breakdown of peptide bond of proteins (proteases) Like other proteins, enzymes behave as ampholytes in an electric field. The isoelectric point for various enzymes are determined. Enzymes like other proteins undergo denaturation and lose enzymatic activity Many purified enzymes, on injection into animal body produce specific antibodies.

Nature of Enzymes.. Contd… 2. They are high molecular weight compounds (10 to 2,000 KDa) They are huge in comparison to substrate Due to large size, posses low rate of diffusion and form colloidal systems in water. 3. They can be precipitated with salts, solvents and other reagents 4. Have high catalytic power. No side products are produced. Do not undergo any change. So small quantity of enzyme can catalyze large quantity of substrate

Catalytic power of an enzyme is measured by the ‘turn over number’ It is defined as: the number of substrate molecules converted into products per unit time, when the enzyme is fully saturated with substrate A single molecule of catalase can convert 50 lakhs H2O2 molecules to H2O in a minute 36 million carbonic acid molecules are produced per minute (60000/sec) by carbonic anhydrase

Turn over number per second Turn over number of some enzymes Enzyme Turn over number per second Lysozyme 0.5 Tryptophan synthetase 2 DNA Polymerase-1 15 Phosphoglucomutase 20.5 B-Galactosidase 208 Lactate Dehydrogenase 1000 B-amylase 18,333

5. Highly specific. They may act On one specific type of substrate molecules Carbonic anhydrase acts only on CO2 and H2O On a group of structurally related compounds LDH converts pyr to lactate and vice versa On only one of the two optical isomers of a compound L-amino acid oxidases On only one of the two geometrical isomers (cis and trans) Fumarase converts fumaric (trans) to L-malic acid . It does not act on cis form of fumaric acid 6. Regulated by naturally occurring compounds

7. Enzymes are sensitive to heat 7. Enzymes are sensitive to heat. Rate of reaction increased to 2-3 times for every 10°C: temperature quotient (Q10) Above 60°C it denatures 8. Enzymes show pH sensitivity (pepsin 1.5, sucrase 6.2, arginase 10)

Enzyme Vs Inorganic Catalysts Enzymes Inorganic Catalysts Protein in nature High catalytic power, very efficient High specificity for substrate and reaction Often 1 Enz, 1 Substrate Regulation by naturally occurring compounds Do not require extreme temp and pressure for reaction Often works at body temp Require extreme temp and pressure Eg: Haber Process for Ammonia synthesis from N and H: t= 700-900K; p= 100-900 atm Most enzymes accelerates reaction by a million times Carbonic anhydrase accelerates 107 times faster than uncatalysed reaction

Enzyme Vs Inorganic Catalysts Enzymes Inorganic Catalysts They can bind so many substrates in short period of time. So small amount of enzyme is needed compared to quantity of substrate. Require coenzyme/ cofactors No such requirements Activity of enzymes are regulated in vivo Activity of enzyme can be increased by activators (mg2+ for kinases) Activity of enzyme can be decreased by inhibitors Factors like temp, pH, substrate concentration and enzyme concentration can affect enzyme activity Enzyme produce only the expected products from a reaction; no side reactions

Similarities: Enzyme & Inorganic Catalysts They are neither consumed nor produced during the course of the reaction They do not cause reaction to take place; they speed up reaction