Enzymes

2. Enzymes Enzymes: Biological catalysts that promote and speed up chemical reactions without themselves being altered (consumed) in the process. They determine the patterns of transformations for chemicals, as well as forms of energy in the living organisms.

3. Historical Events in Discovery of enzymes as biocatalysts-1 • Both enzymology and biochemistry were evolved from the 19th century investigation on the nature of animal digestion and fermentation. • Biochemical reactions could not be reproduced in the lab initially and was thought (e.g., Louis Pasteur) to occur by the action of a “vital force”. • The idea of “catalytic force” or “contact substance” promoting fermentation was introduced in about 1830s. • Addition of alcohol to an aqueous extract of malt (geminating barley) and saliva precipitated a flocculent material which liquefied starch paste and converted it into sugar, this material was named diastase(1833) (later amylase).

4. Enzymes as the biocatalysts-2 • Pepsin was discovered as the active principle in the acid extract of gastric mucosa causing the dissolution of coagulated egg white (1834). • Other “soluble ferments” discovered in the 19th century include trypsin (1857), invertin (later invertase and sucrase, 1864), papain (“vegetable trypsin”, 1879), etc. • “Enzyme (something in yeast) ” was first coined for such “unorganized ferments” by Kühne in 1876. • Enzymes for alcoholic fermentation were found to be active in cell free extracts of yeast (1897, Eduard Buchner): fermentation is a chemical process, not a “vital process”.

5. Enzyme specificity was revealed by studying sugar conversion • Sugars of known structure were synthesized and used as substrates of enzymes. • The a-methylglucoside was found to be hydrolyzed by invertin, but not by emulsin, whereas the b-methylglucoside was cleaved by emulsin, but not by invertin: the enzyme and the glucoside was considered to fit (complement) each other like a lock and a key.

6. Enzymes as the biocatalysts (3) • Relationship of initial velocity (V0) and substrate concentration (S) was examined. • A mathematical description was established for the kinetics of enzyme action (Michaelis and Menten, 1913). • Weak-bonding interactions between the enzymes and their substrates were proposed to distort the substrate and catalyze a reaction (Haldane, 1930s). Before it was known that enzymes are proteins!!! John Burdon Sanderson Haldane (1892-1964) Leonor Michaelis (1875-1949) Maud Menten (1879-1960) A British Geneticist A German A Canadian

7. Formation of an enzyme-substrate (ES) complex was suggested • The activity of invertase in the presence of sucrose survives a temperature that completely destroys it if the sucrose is not present (C O’Sullivan and F. W. Tompson, 1890). • Emil Fisher’s study on the specifity of invertase (1894). • The rate of fermentation of sucrose in the presence of yeast seemed to be independent of the amount of sucrose present, but on the amount of the enzyme (A. J. Brown, 1902). • The kinetics of enzyme action was originally studied using invertase (a hyperbola when V0 was plotted against [S]). • The enzyme (E) was thus assumed to form a complex. (ES) with the substrate (S) before the catalysis.

8. The kinetics of the enzyme-catalyzed reaction were found to be rather different from those of a typical chemical reaction The rate is proportional to the concentration of the reactant in a typical chemical reaction. Enzymes however showed a saturation kinetics: formation of ES complex was hypothesized (1902).

9. Enzymes were found to be proteins • The question of homogeneity of the enzyme preparations frustrated the field of enzymology for many decades. • Nitrogen content analysis and various color tests (for proteins) led to contradictory results. • Filterable coenzymes (co-ferments) were discovered in Buchner’s zymase (Harden and Young, 1906). • Enzymes were thought to be small reactive molecules adsorbed on inactive colloidal material, including proteins ( as by R. Willstätter in the 1920s). • Urease (1926, Sumner) and pepsin (1930, Northrop) were crystallized and found to be solely made of proteins.

10. Urease crystals Sumner, J. B. (1926) “ The isolation and crystallization of the enzyme urease” J. Biol. Chem. 69:435-441.

11. Pepsin crystals Northrop, J. H. (1930) “Crystallin pepsin, 1: Isolation and tests of purity” J. Gen . Physiol. 13:739-766.

13. Not all enzymes are proteins: Some RNA molecules (ribozymes) were found to be catalytic (Sidney Altman and Thomas Cech, 1989).Ribozymes are found to promote RNA processing. Sidney Altman visiting PKU

14. The enzyme theory of life was formulated • Enzymes are central to every biochemical process (Hofmeister, 1901): “life is short and thus has to be catalyzed”. • Isolation, purification and physico-chemical characterization of enzymes would be important for understanding the nature of life. • Without catalysis, the chemical reactions needed to sustain life could not occur on a useful time scale. • Self replication and catalysis are believed to be the two fundamental conditions for life to be evolved. (RNA is thus proposed to be the type of life molecules first evolved).

15. Rate enhancement 2 H2O2→ 2 H2O + O2 Fe3+ →1000 fold Hemoglobin → 1 ,000,000 fold Catalase → 1 ,000,000,000 fold 200,000 catalytic events/second/subunit (near the diffusion-controlled limit). The reaction is sped up by a billion fold! (a prosthetic group) Active site (tetramers)

16. The current understanding on the general features of enzymes Extraordinarily powerful; Highly specific; Be often regulated.

17. Trival name • Gives no idea of source, function or reaction catalyzed by the enzyme. • Example: trypsin, thrombin, pepsin.

18. Systematic Name • According to the International union Of Biochemistry an enzyme name has two parts: -First part is the name of the substrates for the enzyme. -Second part is the type of reaction catalyzed by the enzyme.This part ends with the suffix “ase”. Example: Lactate dehydrogenase

19. EC number Enzymes are classified into six different groups according to the reaction being catalyzed. The nomenclature was determined by the Enzyme Commission in 1961 (with the latest update having occurred in 1992), hence all enzymes are assigned an “EC” number. The classification does not take into account amino acid sequence (ie, homology), protein structure, or chemical mechanism.

20. EC numbers • EC numbers are four digits, for example a.b.c.d, where “a” is the class, “b” is the subclass, “c” is the sub-subclass, and “d” is the sub-sub-subclass. The “b” and “c” digits describe the reaction, while the “d” digit is used to distinguish between different enzymes of the same function based on the actual substrate in the reaction. • Example: for Alcohol:NAD+oxidoreductase EC number is 1.1.1.1

21. The Six Classes • EC 1. Oxidoreductases • EC 2. Transferases • EC 3. Hydrolases • EC 4. Lyases • EC 5. Isomerases • EC 6. Ligases • Additional information on the sub-subclasses and sub-sub-subclasses (i.e, full enzyme classification and names) can be found at the referenced web link. • From the Web version, http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html

22. EC 1. Oxidoreductases • EC 1. Oxidoreductases :catalyze the transfer of hydrogen or oxygen atoms or electrons from one substrate to another, also called oxidases, dehydrogenases, or reductases. Note that since these are ‘redox’ reactions, an electron donor/acceptor is also required to complete the reaction.

23. EC 2. Transferases • EC 2. Transferases – catalyze group transfer reactions, excluding oxidoreductases (which transfer hydrogen or oxygen and are EC 1). These are of the general form: • A-X + B ↔ BX + A

24. EC 3. Hydrolases • EC 3. Hydrolases – catalyze hydrolytic reactions. Includes lipases, esterases, nitrilases, peptidases/proteases. These are of the general form: • A-X + H2O ↔ X-OH + HA

25. EC 4. Lyases • EC 4. Lyases – catalyze non-hydrolytic (covered in EC 3) removal of functional groups from substrates, often creating a double bond in the product; or the reverse reaction, ie, addition of function groups across a double bond. • A-B → A=B + X-Y X Y • Includes decarboxylases and aldolases in the removal direction, and synthases in the addition direction.

26. EC 5. Isomerases • EC 5. Isomerases – catalyzes isomerization reactions, including racemizations and cis-tran isomerizations.

27. EC 6. Ligases • EC 6. Ligases -- catalyzes the synthesis of various (mostly C-X) bonds, coupled with the breakdown of energy-containing substrates, usually ATP

28. Mechanism