Enzymic formation

Enzyme Formation and Polysaccharide Synthesis by Bacteria. III. Polysaccharides Produced by Nitrogen-fixing Organisms, W. A. Cooper, W. D. Daker, and M. Stacey, Biochem. J., 32 (1938) 1752-1758. 

In 1933, Schlubach and Knoop32 isolated a di-D-fructose dianhydride from Jerusalem artichoke and tentatively identified it as difructose anhydride I [a-D-Fru/-1,2 2,1 - 3-D-Fn / (5)]. Alliuminoside ( -D-fructofuranose- -D-fructofura-nose 2,6 6,2 -dianhydride) was isolated from tubers of Allium sewertzowi by Strepkov33 in 1958. Uchiyama34 has demonstrated the enzymic formation of a-D-Fru/-1,2 2,3 -(3-D-Fru/ [di-D-fructose anhydride III (6)] from inulin by a homogenate of the roots of Lycoris radiata Herbert. 

It is possible to use isolated, partially purified enzymes (dehydrogenases) for the reduction of ketones to optically active secondary alcohols. However, a different set of complications arises. The new C H bond is formed by delivery of the hydrogen atom from an enzyme cofactor, nicotinamide adenine dinucleotide (phosphate) NAD(P) in its reduced form. The cofactor is too expensive to be used in a stoichiometric quantity and must be recycled in situ. Recycling methods are relatively simple, using a sacrificial alcohol, or a second enzyme (formate dehydrogenase is popular) but the real and apparent complexity of the ensuing process (Scheme 8)[331 provides too much of a disincentive to investigation by non-experts. 

K17. Kurahashi, K., Enzyme formation in galactose-negative mutants of Escherichia coll. Science 125, 114-116 (1957). 

Franklin MR. The enzymic formation of methylenedioxyphenyl derivative exhibiting an isocyanide-like spectrum with reduced cytochrome P-450 in hepatic microsomes. Xenobiot-ica 1971 1(6) 581—591. 

Boyer, P.D. Segal, H.L. (1954). Sulfhydryl groups and glyceraldehyde-3-phosphate dehydrogenase and acyl-enzyme formation. In Metabolic Pathways. (Greenberg, D.M., Ed.), 2nd ed., Vol. 1, pp. 520-532. Academic Press, New York. 

J. R. Alvarez-Idaboy, R. Gonzalez-Jonte, A. Hernandez-Laguna, Y. G. Smeyers, Reaction Mechanism of the Acyl-Enzyme Formation in /3-Lactam Hydrolysis by Means of Quantum Chemical Modeling , J. Mol. Struct. 2000, 204, 13 - 28. 

One of the important consequences of studying catalysis by mutant enzymes in comparison with wild-type enzymes is the possibility of identifying residues involved in catalysis that are not apparent from crystal structure determinations. This has been usefully applied (Fersht et al., 1988) to the tyrosine activation step in tyrosine tRNA synthetase (47) and (49). The residues Lys-82, Arg-86, Lys-230 and Lys-233 were replaced by alanine. Each mutation was studied in turn, and comparison with the wild-type enzyme revealed that each mutant was substantially less effective in catalysing formation of tyrosyl adenylate. Kinetic studies showed that these residues interact with the transition state for formation of tyrosyl adenylate and pyrophosphate from tyrosine and ATP and have relatively minor effects on the binding of tyrosine and tyrosyl adenylate. However, the crystal structures of the tyrosine-enzyme complex (Brick and Blow, 1987) and tyrosyl adenylate complex (Rubin and Blow, 1981) show that the residues Lys-82 and Arg-86 are on one side of the substrate-binding site and Lys-230 and Lys-233 are on the opposite side. It would be concluded from the crystal structures that not all four residues could be simultaneously involved in the catalytic process. Movement of one pair of residues close to the substrate moves the other pair of residues away. It is therefore concluded from the kinetic effects observed for the mutants that, in the wild-type enzyme, formation of the transition state for the reaction involves a conformational change to a structure which differs from the enzyme structure in the complex with tyrosine or tyrosine adenylate. The induced fit to the transition-state structure must allow interaction with all four residues simultaneously. 

N-Carbobenzoxy-L-alanine-/>-nitrophenyl ester is a specific substrate for elastase in which the rate-limiting step is deacylation, that is, hydrolysis of the acyl-enzyme intermediate. In 70% methanol over a reasonable temperature range the energy of activation of the turnover reaction, that is, deacylation, is 15.4 kcal mol. In the pH 6-7 region in this cryoprotective solvent, the turnover reacdon can be made negligibly slow at temperatures of -50 C or below. Under such conditions/i-nitro-phenol is released concurrent to acyl enzyme formation in a 1 1 stoichiometry with active enzyme in the presence of excess substrate. In other words, even at low temperatures, the acylation rate is much faster than deacylation and the acyl enzyme will accumulate on the enzyme. The rate of acyl-enzyme formation can be monitored by following the rate of p-nitrophenol release, and thus the concentration of trapped acyl enzyme may be determined. This calculadon has been carried out and... 

The donor types D3, D4, and D6 of Keilin and Nicholls (37) all reduce compound I of Type A enzymes directly to the ferric state in a two-electron process without detectable intermediates. Each of these donors is probably also able to bind in the heme pocket of the free enzyme. Alcohols (type D3) form complexes with free ferric Type A enzymes whose apparent affinities parallel the effectiveness of the same alcohols as compound I donors (39). Formate (type D3) reacts with mammalian ferric enzyme at a rate identical to the rate with which it reduces compound I to free enz5mie (22). Its oxidation by compound I may thus share an initial step analogous to its complex formation with ferric enzyme. Formate also catalyzes the reduction of compound II to ferric enzyme by endogenous donors in the enz5mie (40, 41). Both compound I and compound II may thus share with the free enzyme the ability to ligate formate in the heme pocket. Nitrite, which is oxidized to nitrate by a two-electron reaction with compoimd I (type D4), also forms a characteristic complex with free enzyme (42). In both cases the reaction involves the donor in its protonated (HNO2) form. 

Enzymic formation of theobromine from 7-mehylxanthine and of caffeine from theobromine. Biochem J 1975 ... 

More complex reductions of CO2 by enzyme cascades have also been achieved. A combination of an electron mediator and two enzymes, formate dehydrogenase and methanol dehydrogenase, was used to reduce CO2 to methanol. This system operates with current efficiencies as high as 90% and low overpotentials (approximately —0.8 V vs. SCE at pH 7) [125]. The high selectivity and energy efficiency of this system indicate the potential of enzyme cascades. There are also drawbacks to these systems. In general, enzymes are... 

Interestingly the Mo enzyme formate dehydrogenase shows an EXAFS pattern that has been interpreted in terms of approximately three Mo—0 = 1.74 A distances indicating the likely presence of an Mo03 unit bound to non-sulfur ligands.69... 

The reversible, enzymic formation of this D-mannolipid involves the transfer of the D-mannosyl group from GDP-D-mannose to undecaprenyl... 

Nishizuka, Y. and Hayaishi, 0.1962. Enzymic formation of lactobionic acid from lactose. J. Biol. Chem. 237, 2721-2728. 

Like inorganic phosphate, inorganic sulfate can be converted into an activated form in which the sulfate resembles the terminal phospho group of ATP (Eq. 17-38). The resulting activated sulfo group can be transferred to other compounds including enzymes. Formation of... 

In a very similar way, acyl-enzyme formation depends upon the unsaturation level of the chains, and upon the position of the double bond(s) on the chains. Geotrichum candidum lipase is known to prefer fatty acids with a double bond on the C9. like oleic acid. 

V. Enzymic Formation of Pyrrolidone Carboxylic Acid from... 

The formation of pyrrolidone carboxylic acid under these conditions has been interpreted to indicate the enzymic formation of an activated glutamate intermediate. The available evidence supports the view that glutamate and ATP interact on the active site of the enzyme to yield en-... 

Other work showed that active hybrids (38, 74) were obtained in vitro by mixing the monomers of two pseudo-revertants of E. coli that produce active enzymes which are electrophoretically different from the wild-type enzyme. Also, complimentation studies with E. coli mutants showed in vivo hybrid enzyme formation (75). 

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