Enzymology

Holzer, Biochem. Biophys. Res. Comm., 1966, 

Dihydroxyacetone phosphate (82) is a substrate for a-glycero-phosphate dehydrogenase, aldolase, and triose phosphate isomerase, and its O-alkyl ethers are intermediates in the biosynthesis of phospholipids. In neutral aqueous solution at 20 °C, dihydroxyacetone phosphate exists as an equilibrium mixture of the keto (82), gem-d o (83), and enol (84) forms, as shown by n.m.r. spectroscopy. The proportion of (82) to (83) 

Compartmentation and Regulation of Starch Synthesis and Degradation in Chloroplasts 

Sucrose-P synthase (SPS) activity in spinach is regulated at three levels, protein quantity, protein phosphorylation/dephosphorylation, and allosteric control.188,189 

Methanol disproportionation involves the reduction of 3 mol CH3OH to CH4 with 6[H] derived from the oxidation of 1 mol CH3OH to CO2  

Free energy changes (AG ) and redox potentials (E° ) of partial reactions involved in CH3OH 

Methanol disproportionation includes two sites of A/xNa generation (1) Methanol reduction generates a secondary AftNa from a primary A/IH by the activity of the Na /H antiporter. This was concluded from the finding that CH4 formation from H2/CH3OH in Methanosarcina barkeri was coupled with Na extrusion, which was sensitive to Na /H antiporter inhibitors and protonophores [108]. (2) The oxidation of methylene-H4MPT to CO2 and 4[H] is coupled with the generation of a primary A/INa as indicated from the fact that Na translocation associated with formaldehyde conversion to CO2 and 2H2 was not sensitive towards Na /H antiporter inhibitors and protonophores [105]. 

The eNOS and iNOS are homodimer hemproteins with the molecular weight of 130-135 kDa per subunit. The subcellular localization of eNOS and iNOS appears to be different 

The equilibrium position of this reaction is 6 X 10 which is quite unfavorable for the synthesis of acetoacetyl-CoA. However, as will be pointed out below, when it functions in conjunction with the next enzyme in the sequence, synthesis is favored. The turnover number in the forward direction (as written) was found to be 1770 while that of the reverse was 54000. The enzyme from mitochondria has two electrophoretically distinct forms, and the cytoplasmic enzyme could be clearly distinguished from the two mitochondrial proteins by the difference in their isoelectric points. Middleton surveyed many tissues from rat as well as selected tissues from ox and pigeon for both the cytoplasmic and the mitochondrial enzymes. The highest levels of the cytoplasmic enzyme were found in the liver, adrenal, brciin of the neonate, and ileum [14,15]. There was an obvious positive correlation between sterol biosynthetic capacity and the distribution of this enzyme. The mitochondrial enzyme was found predominantly in heart, kidney, and liver. The cytosolic enzyme has also been highly purified from rat liver [16]. 

The enzyme could be inactivated by NaBH4 in the presence of either acetoacetyl-CoA or acetyl-CoA. This observation strongly suggests that the reaction is through a Claisen condensation with an amine as the base and with an enzyme substrate ketimine as an intermediate [20]. A mechanism was postulated for the reaction as indicated in Fig. 2. 

The use of paramagnetic probes in magnetic resonance studies on phosphoryl transfer enzymes, e.g. creatine kinase, has been reviewed, and model reactions with phosphoroguanidates have led to new ideas on the mechanism of action of this enzyme. The pH-rate profile for the 

A phosphoryl-enzyme intermediate is formed during reactions catalysed by alkaline phosphatase. 0-4-Nitrophenyl phosphorothioate is hydrolysed 1000 times more slowly by alkaline phosphatase than is its oxygen analogue, suggesting an 5n2(P) mechanism for the phosphorylation of the enzyme. From a kinetic study of the reaction between a series of alkyl-, aryl-, and arylamido-phosphates and alkaline phosphatase, it has been shown that steric factors are important in these reactions. Moreover, although amidophosphates [e.g. (69)] are substrates for this enzyme  

Under mild conditions, chloro- and bromo-acetol phosphates (70), reactive analogues of dihydroxyacetone phosphate, inactivate yeast fructose 

The effect of a number of oximes derived from pyridines on rat-brain acetyl cholinesterase which had been inactivated by isopropyl methyl-phosphonofluoridate has been studied, the most effective at restoring enzymic activity being (75). Acetyl cholinesterase is also inhibited by aryl 

Infrared spectroscopy has been used advantageously in studies of enzymatic reactions. Chapter 11 contains a discussion of the application of the infrared method of studying hydrogen-deuterium exchange in various enzyme molecules, and the application of this method for studying the effects of substrates and of inhibitor substances on the conformations of the enzymes. 

This chapter gives some examples of how infrared measurements have been applied to studies of mechanisms of enzyme reactions, stereospecificity of reactions, inhibition of reactions, kinetics, properties of substrates, characterization of products of reactions, and identification of organisms by the kinds of enzymatic action displayed by them. 

The vast majority of cytochrome P450 monooxygenases catalyze the reductive scission of dioxygen, which requires the consecutive delivery of two electrons to the heme iron. P450s utilize reducing equivalents (electrons in the form of hydride ions) ultimately derived from the pyridine cofactors NADH or NADPH and transferred to the heme via special redox proteins [38, 39]. 

Some P450s are able to catalyze oxidative phenol coupling, a reaction usually carried out by peroxidases. Three independent P450 monooxygenases with such activity have been shown to be involved in the synthesis of vancomycin-type antibiotics in Amycolatopsis balhimycina [67]. 

Many other unusual types of oxidative and also some reductive reactions catalyzed by P450s have been described in the literature, including oxidative deamination, desulfurylation, oxidative dehalogenation, isomerization, dehydrogenation, dehydration, reductive dehalogenation, epoxide reduction, and others [54, 57, 70]. 

The branched-chain amino acids valine and leucine are synthesized from pyruvate while isoleucine is derived from one molecule of pyruvate and one molecule of a-ketobutyrate. The initial pathway enzyme for all 

---

Marriott G (ed) 1998 Caged Compounds (Methods in Enzymology 291) (New York Academic)... 

P66 P.E. Bourne, H.M. Berman, B. McMahon, K.D. Watenpaugh, J.D. Westbrook, P.M.D. Fitzgerald, Macromole-cular crystallographic information file, in Methods in Enzymology 1997, 277, 571-590. 

Chou P Y and G D Fasman 1978. Prediction of the Secondary Structure of Proteins from Tlieir Amino Acid Sequence. Advances in Enzymology 47 45-148. 

Pearson W R 1990, Rapid and Sensitive Sequence Comparison with FASTP and FASTA. Methoc Enzymology 183 63-98. 

Industrial asphalts Industrial catalysts Industrial coatings Industrial enzymology Industrial ethanol Industrial furnaces Industrial gas Industrial hygiene... 

Industrial appHcations of enzymology form an important branch of biotechnology. Enzymatic processes enable natural raw materials to be upgraded and turned into finished products. They offer alternative ways of making products previously made only by conventional chemical processes. 

The development of the submerged fermentation technique resulted in tremendous progress in the field of industrial enzymology. This technique was used in the early 1950s for the production of bacterial amylases for the textile industry. 

Perhaps the biggest impact on the practical utilization of enzymes has been the development of nonaqueous enzymology (11,16,33,35). The use of enzymes in nonaqueous media gready expands the scope of suitable transformations, simplifies thek use, and enhances stabiUty. It also provides an easy means of regulation of the substrate specificity and regio- and enantioselectivity of enzymes by changing the reaction medium. 

This chapter lists some representative examples of biochemicals and their origins, a brief indication of key techniques used in their purification, and literature references where further details may be found. Simpler low molecular weight compounds, particularly those that may have been prepared by chemical syntheses, e.g. acetic acid, glycine, will be found in Chapter 4. Only a small number of enzymes and proteins are included because of space limitations. The purification of some of the ones that have been included has been described only briefly. The reader is referred to comprehensive texts such as the Methods Enzymol (Academic Press) series which currently runs to more than 344 volumes and The Enzymes (3rd Edn, Academic Press) which runs to 22 volumes for methods of preparation and purification of proteins and enzymes. Leading referenees on proteins will be found in Advances in Protein Chemistry (59 volumes. Academic Press) and on enzymes will be found in Advances in Enzymology (72 volumes, then became Advances in Enzymology and Related Area of Molecular Biology, J Wiley Sons). The Annual Review of Biochemistry (Annual Review Inc. Patio Alto California) also is an excellent source of key references to the up-to-date information on known and new natural compounds, from small molecules, e.g. enzyme cofactors to proteins and nucleic acids. 

Tucker, P.W., Hazen, E.E., Colton, F.A. Staphylococcal nuclease reviewed a prototypic study in contemporary enzymology. III. Correlation of fhe three-dimensional structure with the mechanisms of enzymatic action. Mol. Cell. Biochem. 

T. C. Bruice and S. I Benkovic, Bioorganic Mechanisms, Vol. 1, W. A. Benjamin, New brk, 1966, pp. 1-258 W. P. Jencks, Catalysis in Chemistry and Enzymology, McGraw-Hill, New York, 1969 M. L. Bender, Mechanisms of Homogeneous Catalysis from Protons to Proteins, Wiley-Interscience, New York, 1971 C. Walsh, Enzymatic Reaction Mechanisms, W. H. Freeman, San Francisco, 1979 A. Fersht, Enzyme Structure and Mechanism, 2nd ed., W. H. Freeman, New York, 1985. 

Jencks, W.P. "Catalysis in Chemistry and Enzymology McGraw-Hill New York, 1969. 

Dentscher, M. P., ed., 1990. Guide to Protein Purification. Vol. 182, Methods in Enzymology. San Diego Academic Press, 894 pp. 

Karger, B. L., and Fiancock, W. S., eds. 1996. Methods in Enzymology 271, Section III Protein Structure Analysis by Mass Sj ectrometry, f R. Yates P tide Characterization by Mass Spectrometry, B. L. Gillece-Castro and J. T. Stnlts. New York Academic Pre.s.s. 

Towler, D. A., Gordon, J. L, Adams, S. R, and Glaser, L., 1988. The biology and enzymology of eukaryodc protein acylation. Annual Review of Biochemistry 57 69—99. 

Wn, R., 1993. Development of enzyme-ba.sed mediods for DNA. sequence analy.sis and dieir application in genome projects. Methods in Enzymology 67 431-468. 

AdapLed. from Uhleii, M., and Moles, T., 1990. Gene fusions for purpose of expression An inu oducdon. Methods in Enzymology 185 129-143. 

Berger, S. L., and Kimmel, A. R., eds., 1987. Guide to Molecular Cloning Techniques. Methods in Enzymology, Volume 152. New York Academic Press. 

Silverman, R. B., 1988. Mechanism-Based Enzyme Inactivation Chemistry and Enzymology, Vols. I and II. Boca Raton, FL CRC Press. 

Radzicka, A., and Wolfenden, R., 1995. Transition state and mnltisnbstrate analog inhibitors. Methods in Enzymology 249 284-312. 

Jencks, W. P., 1969. Catalysis in Chemistry and Enzymology. New York McGraw-Hill. 

Fruton, J., 1976. The mechanism of die catalytic action of pepsin and related acid proteina.ses. Advances in Enzymology 44 1-36. 

The versatile chemistry of pyridoxal phosphate offers a rich learning experience for the student of mechanistic chemistry. William Jencks, in his classic text. Catalysis in Chemistry and Enzymology, writes ... 

Miziorko, H. M., and Lorimer, G. H., 1983. Ribnlose-l,5-bisphosphate car-boxylase/oxygenase. Annual Review of Biochemistry 52 507-535. An early review of die enzymological properties of rnbisco. 

Earner, J., 1990. Insulin and the stimulation of glycogen synthesis The road from glycogen structure to glycogen synthase to cyclic AMP-dependent protein kinase to insulin mediators. Advances in Enzymology 63 173-231. 

Sukalski, K. A., and Nordlie, R. C., 1989. Glucose-6-phosphatase Two concepts of membrane-funcdon relationship. Advances in Enzymology 62 93-117. 

Enzymology and molecular biology of aflatoxin biosynthesis 97CRV2537. 

---