Chemistry PAPER No. 16: BIO-ORGANIC AND BIOPHYSICAL CHEMISTRY MODULE No. 8: Introduction to Enzymes, their nomenclature & classification

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1 Subject Chemistry Paper No and Title Module No and Title Module Tag 16; Bioorganic and Biophysical Chemistry 8; Introduction to Enzymes, their and nomenclature CHE_P16_M8

2 TABLE OF CONTENTS 1. Learning Outcomes 2. Introduction 3. Introduction to Enzymes 3.1 Enzymes are biocatalysts 3.2 History of Enzymology 3.3 Interaction of Enzyme with substrate 3.4 Cofactors and coenzymes 4. Nomenclature and of enzymes 4.1 Common names 4.2 IUBMB and nomenclature 5. Summary

3 1. Learning Outcomes After studying this module, you shall be able to: Know what are enzymes and how they are different from chemical catalysts Learn history f discovery of enzymes How to enzymes recognize their substrates? Know enzymes require cofactors and coenzymes beyond amino acids which it is made of. Learn how enzymes are named. 2. Introduction Enzymes are biological catalysts Mostly enzymes are proteins which possess catalytic activity. Enzymes were discovered in 18 th century. They recognize their specific substrate and bind to them via a cleft termed as substrate binding site. Once they bind the substrate, they carry out the reaction and release products. For this binding, they require structural as well as electronic complementarity with the substrate. They mediate the reactions at physiological ph and temperature at rates much higher than the uncatalyzed reactions. Some enzymes carry out catalysis by virtue of amino acids that they are made of, while some require additional cofactors and coenzymes for full catalytic activity. Enzymes are named by four digit nomenclature as well as common names. This chapter also discusses the history of enzymology. 3. Introduction to Enzymes 3.1 Enzymes are biocatalysts The term enzyme was coined by Wilhelm Friedrich Kuhne in Enzymes are biological catalysts that differ from chemical catalysts in following features: (a) They work milder reaction conditions unlike chemical catalysts. Enzymes work physiological temperature and ph for their activity. (b) Enzymes have higher reaction specificity. They bind to specific substrate, carry the catalytic reaction and release specific products. (c) Enzymes enhance the rates of reaction they catalyze by several orders of magnitude when compared with uncatalysed reactions. The rates of enzyme catalyzed reactions are much higher than the reactions catalyzed by chemical catalysts.

4 Table 1: Enzyme enhance the rates of reactions compared to uncatalyzed reactions. Name of the Enzyme Fold enhancement in rate Carboxypeptidase A Urease Triose phosphate isomerase 10 9 (d) Enzyme catalyzed reactions are well regulated. These reaction rates are not only influenced by substrate or product concentrations but also can be regulated by covalent modifications of enzymes etc. Most enzymes are proteinecous in nature but RNA enzymes also exist. RNA enzymes are ribozymes. 3.2 History of Enzymology Research work on fermentation by Joseph Gay Lussac determined that yeast decomposes sugar into carbon dioxide and ethanol. Jacob Berzelius proposed that the malt extract (diastase) catalyzes starch hydrolysis much efficiently than chemical catalyst H 2 SO 4. However, in mid nineteenth century, Louis Pasteur proposed that fermentation can only take place in living cells. The term enzyme was coined by W.H. Kuhne in Eduard Buchner in 1897 showed fermentation in cell free extracts illustrating that fermentation can occur outside the living cells. He named the enzyme responsible for fermentation of sucrose zymase. In 1907, he received Nobel Prize in Chemistry for his pioneer work on discovery of cell free fermentation. In 1926, James Sumner gave identity to the enzymes. His work on jack bean urease (which catalyzes hydrolysis of urea to ammonia and carbon dioxide) proved that enzymes are pure proteins. He crystallized urease. The crystals consisted entirely of proteins. But his work was not accepted till John Northrop and Moses Kunitz showed correlation of activities of enzymes pepsin, trypsin and chymotrypsin with the amount of protein. In 1946, these three scientists were then awarded Nobel Prize in Chemistry. In 1963, the amino acid sequence of the first enzyme bovie pancreatic ribonuclease A was given and in 1965, the first enzyme whose X-ray structure was worked out was that of hen egg white lysozyme by David Phillips. 3.3 Interaction of Enzyme with Substrate

5 3.3.1 Geometric and Electronic complementarity Van der Waals forces, hydrophobic interactions and H-bonding are the noncovalent forces driving interactions between substrate and product. Substrate binding site in enzyme is a cleft in the enzyme in which the substrate fits. This is called the geometric or physical complementarity (Figure 1). Figure 1. Depiction of geometric and electronic complementarity between substrate and enzyme. The amino acids that form the substrate binding site of the enzyme form attractive interaction with the substrate. This is termed as electronic complementarity (Figure 1) Models of substrate binding to enzyme Two models have been proposed for substrate binding to enzyme: (a) Lock and key model In 1894, Emil Fischer proposed that both enzyme and substrate possess geometric shapes complementary to each other such that substrate perfectly fits the substrate binding site of the enzyme just like a key fits into the lock. Most importantly, enzyme possesses the substrate binding site even in the absence of the substrate.

6 A B Figure 2. (A) The lock and key model of substrate binding to enzyme. (B) induced fit model. (b) Induced fit model Induced fit model was proposed by Daniel Koshland in He suggested that the substrate binding site is not rigid but reshapes or moulds itself after initial interaction with the substrate (Figure 2B) to bind the substrate perfectly. 3.4 Cofactors and Coenzymes Most enzymes are proteins composed entirely of amino acids. Few enzymes require additional chemical moieties other than amino acids. These chemical moieties are termed as cofactors. For example: Hexokinase requires Mg 2+ for its catalytic activity. These cofactors can be inorganic or organic moieties. If they are organic in nature, they are termed as coenzymes. These coenzymes are derived from vitamins. Few examples of cofactors and coenzymes have been listed in Table 1. Cofactor/ Coenzymes Mg 2+ Zn 2+ Ni 2+ Biotin (Coenzyme form-biocytin) Vitamin B 2 / Riboflavin (Flavin adenine dinucleotide) Vitamin B 12 (Coenzyme B 12 ) Vitamin B 1 /Thiamin (Thiamine pyrophosphate) Enzymes Hexokinase Carbonic anhydrase Urease Pyruvate carboxylase Succinate dehydrogenase Methionine synthase Pyruvate dehydrogenase

7 Some enzymes can require both metal ions and coenzymes for their activity. If the metal ion or coenzyme is tightly bound to the enzyme, it is termed as prosthetic group. The enzyme without the prosthetic group is called the apoenzyme. Along with the prosthetic group, it is called holoenzyme. Holoenzyme is thus the complete catalytically active form of the enzyme. Holoenzyme Apoenzyme + cofactor 4. Nomenclature and Classification of Enzymes 4.1 Common names The enzymes are named by adding suffix -ase to the name of the substrate or their activity. For example: Hexokinase mediates phosphorylation of hexoses; Urease catalyzes the hydrolysis of urea. RNA polymerase catalyzes the polymerization of ribonucleotides to form RNA. 4.2 IUBMB and nomenclature With the increasing number of enzymes, common names became less popular to avoid same names of two enzymes and one enzyme carrying different names. Hence, to avoid this confusion, International Union of Biochemistry and Molecular Biology (IUBMB) adopted a systematic and nomenclature of enymes. According to this, enzymes are functionally classified into six classes (Table 3). Class 1 belongs to all the oxidoreductases, Class 2 to transferases and so on. These classes have been subdivided into subclasses and subsubclasses. Each enzyme has been allotted two names and four digit. For example: EC ; where EC stands for enzyme commission, the first digit (3) stands for the class to which enzyme belongs i.e. hydrolase (Table 3), the second digit (4) stands for subclass peptide hydrolases (Table 4), the third digit (17) belongs to sub-subclass metallocarboxypeptidases (Table 5), the fourth digit (1) belongs to carboxypeptidase A (Table 6). Thus, this four digit nomenclature describes carboxypeptidase A. Table 3: Enzymes are classified into six different functional classes. Group Reaction catalyzed Typical reaction EC 1 Oxidoreductases EC 2 Transferases To catalyze oxidation/reduction reactions; transfer of H and O atoms or electrons from one substance to another Transfer of a functional group from AH + B A + BH (reduced) A + O AO (oxidized) AB + C A + BC Enzyme example(s) with trivial name Dehydrogenase, oxidase Transaminase, kinase

8 EC 3 Hydrolases EC 4 Lyases EC 5 Isomerases EC 6 Ligases one substance to another. The group may be methyl-, acyl-, amino- or phosphate group Formation of two products from a substrate by hydrolysis Non-hydrolytic addition or removal of groups from substrates. C-C, C-N, C-O or C-S bonds may be cleaved Intramolecule rearrangement, i.e. isomerization changes within a single molecule Join together two molecules by synthesis of new C-O, C-S, C-N or C-C bonds with simultaneous breakdown of ATP AB + H 2 O AOH + BH RCOCOOH RCOH + CO 2 or [X-A-B-Y] [A=B + X-Y] AB BA X + Y+ ATP XY + ADP + Pi Lipase, amylase, peptidase Decarboxylase Isomerase, mutase Synthetase Table 4: The list of subclasses of Class 3 Hydrolases. EC Acting on ester bonds. EC Glycosylases. EC Acting on ether bonds. EC Acting on peptide bonds (peptide hydrolases). EC Acting on carbon-nitrogen bonds, other than peptide bonds. EC Acting on acid anhydrides. EC Acting on carbon-carbon bonds. EC Acting on halide bonds. EC Acting on phosphorus-nitrogen bonds. EC Acting on sulfur-nitrogen bonds. EC Acting on carbon-phosphorus bonds. EC Acting on sulfur-sulfur bonds. EC Acting on carbon-sulfur bonds. Table 5: The list of sub-subclasses of 3.4._._. (Hydrolases acting on peptide bonds)

9 EC Hydrolases. EC Acting on peptide bonds (peptide hydrolases). EC Aminopeptidases. EC Dipeptidases. EC Dipeptidyl-peptidases and tripeptidyl-peptidases. EC Peptidyl-dipeptidases. EC Serine-type carboxypeptidases. EC Metallocarboxypeptidases. EC Cysteine-type carboxypeptidases. EC Omega peptidases. EC Serine endopeptidases. EC Cysteine endopeptidases. EC Aspartic endopeptidases. EC Metalloendopeptidases. EC Threonine endopeptidases. Table 6: The list describing EC _ in IUBMB nomenclature. EC Hydrolases. EC Acting on peptide bonds (peptide hydrolases). EC Metallocarboxypeptidases. EC Carboxypeptidase A. EC Carboxypeptidase B. EC Lysine carboxypeptidase. EC Gly-Xaa carboxypeptidase. EC Alanine carboxypeptidase. EC Muramoylpentapeptide carboxypeptidase. EC Carboxypeptidase E. EC Glutamate carboxypeptidase. EC Carboxypeptidase M. EC Muramoyltetrapeptide carboxypeptidase. EC Zinc D-Ala-D-Ala carboxypeptidase. EC Carboxypeptidase A2. EC Membrane Pro-Xaa carboxypeptidase. EC Tubulinyl-Tyr carboxypeptidase. EC Carboxypeptidase T. EC Carboxypeptidase Taq. EC Carboxypeptidase U. EC Glutamate carboxypeptidase Ii.

10 EC Metallocarboxypeptidase D. EC Angiotensin-converting enzyme 2. EC n1 [CysO]-cysteine peptidase. Table 7. Overview of the first two digits of the four digit nomenclature of Enzymes signifying their class and subclass. Subclass EC 1 Oxidoreductases Name EC 1.1 EC 1.2 EC 1.3 EC 1.4 EC 1.5 EC 1.6 EC 1.7 EC 1.8 EC 1.9 EC 1.10 EC 1.11 EC 1.12 EC 1.13 EC 1.14 EC 1.15 EC 1.16 EC 1.17 EC 1.18 EC 1.19 EC 1.20 EC 1.21 EC 1.22 Acting on the CH-OH group of donors Acting on the aldehyde or oxo group of donors Acting on the CH-CH group of donors Acting on the CH-NH 2 group of donors Acting on the CH-NH group of donors Acting on NADH or NADPH Acting on other nitrogenous compounds as donors Acting on a sulfur group of donors Acting on a heme group of donors Acting on diphenols and related substances as donors Acting on a peroxide as acceptor Acting on hydrogen as donor Acting on single donors with incorporation of molecular oxygen (oxygenases) Acting on paired donors, with incorporation or reduction of molecular oxygen Acting on superoxide radicals as acceptor Oxidising metal ions Acting on CH or CH 2 groups Acting on iron-sulfur proteins as donors Acting on reduced flavodoxin as donor Acting on phosphorus or arsenic in donors Acting on the reaction X-H + Y-H = X-Y Acting on halogen in donors

11 EC 1.23 EC 1.97 EC 2 EC 2.1 EC 2.2 EC 2.3 EC 2.4 EC 2.5 EC 2.6 EC 2.7 EC 2.8 EC 2.9 EC 2.10 EC 3 EC 3.1 EC 3.2 EC 3.3 EC 3.4 EC 3.5 EC 3.6 EC 3.7 EC 3.8 EC 3.9 EC 3.10 EC 3.11 Reducing C-O-C group as acceptor Other oxidoreductases Transferases Transferring one-carbon groups Transferring aldehyde or ketonic groups Acyltransferases Glycosyltransferases Transferring alkyl or aryl groups, other than methyl groups Transferring nitrogenous groups Transferring phosphorus-containing groups Transferring sulfur-containing groups Transferring selenium-containing groups Transferring molybdenum- or tungsten-containing groups Hydrolases Acting on ester bonds Glycosylases Acting on ether bonds Acting on peptide bonds (peptidases) Acting on carbon-nitrogen bonds, other than peptide bonds Acting on acid anhydrides Acting on carbon-carbon bonds Acting on halide bonds Acting on phosphorus-nitrogen bonds Acting on sulfur-nitrogen bonds Acting on carbon-phosphorus bonds EC 3.12 EC 3.13 EC 4 EC 4.1 EC 4.2 EC 4.3 EC 4.4 Acting on sulfur-sulfur bonds Acting on carbon-sulfur bonds Lyases Carbon-carbon lyases Carbon-oxygen lyases Carbon-nitrogen lyases Carbon-sulfur lyases

12 EC 4.5 EC 4.6 EC 4.7 EC 4.99 EC 5 EC 5.1 EC 5.2 EC 5.3 EC 5.4 EC 5.5 EC 5.99 EC 6 EC 6.1 EC 6.2 EC 6.3 EC 6.4 EC 6.5 EC 6.6 Carbon-halide lyases Phosphorus-oxygen lyases Carbon-phosphorus lyases Other lyases Isomerases Racemases and epimerases cis-trans-isomerases Intramolecular isomerases Intramolecular transferases (mutases) Intramolecular lyases Other isomerases Ligases Forming carbon oxygen bonds Forming carbon sulfur bonds Forming carbon nitrogen bonds Forming carbon carbon bonds Forming phosphoric ester bonds Forming nitrogen metal bonds Table 7 presents the summary of the first two digits of the four digit nomenclature of enzymes. ( We just looked at one example to explain four digit nomenclature of enzymes. Lets look at some more common enzymes like trypsin. Its IUBMB nomenclature is For Chymotrypsin, it is ; Hexokinase ; Lysozyme ; Urease Thus, this EC number m.n.o.p are assigned to each enzyme where m represents the class, n is the subclass, o is the subsubclass. n and o describe the reaction catalysed. Lastly, p is the sub-sub-subclass which differentiates two enzymes on the basis of the substrate they use.

13 5. Summary Enzymes are biological catalysts that are proteineous in nature. First enzyme crystallized was jack bean urease by James Sumner. First enzyme whose primary structure was given was Ribonuclease A from bovine pancreas. First enzyme whose X-ray structure was given was lysozyme from hen egg white. Geometric and electronic complementarity exists between enzyme and its substrate. Few enzymes also require cofactors or coenzymes for complete catalytic activity. Enzymes are commonly named by adding a suffix -ase to the reaction they catalyze or named by four digit name specifying class, subclass, details of the reaction they catalyze.