Coenzyme binding

Ohlsson, I., Nordstrom, B., Branden, C.-I. Structural and functional similarities within the coenzyme binding domains of dehydrogenases. /. Mol. Biol. 89 339-354, 1974. 

Catalysis by flavoenzymes has been reviewed and various analogues of FAD have been prepared e.g. P -adenosine-P -riboflavin triphosphate and flavin-nicotinamide dinucleotide ) which show little enzymic activity. The kinetic constants of the interaction between nicotinamide-4-methyl-5-acetylimidazole dinucleotide (39) and lactic dehydrogenase suggest the presence of an anionic group near the adenine residue at the coenzyme binding site of the enzyme. ... 

Non-metals, C, H, N, O, S and P, in the major irreversible non-equilibrated chemistry, in catalysed flow (organic chemistry) covered in Chapter 4 (see Table 5.3) and equilibrated control systems of substrate and coenzyme bindings... 

Figure 4.9 (a) Triose phosphate isomerase (TIM), has a (3-a-(3 structure made up of eight P-a motifs terminating in a final a-helix, which form a barrel-like structure, (b) An open twisted P-sheet with helices on both sides, such as the coenzyme-binding domain of many dehydrogenases. (From Branden and Tooze, 1991. Reproduced by permission of Garland Publishing, Inc.)... 

Such intermediates are known to form between substrate and enzyme in the aldolase reaction and between pyri-doxal 5-phosphate and the amino group of enzyme or substrate in aminotransferase reactions. In the latter case, aldimine formation accounts for the high affinity of coenzyme binding to apotransaminases. 

Some coenzymes bind loosely near the active site of the enzyme and thus act like substrates, while others are covalently bound to the enzyme as a prosthetic group. 

Fig. 2.2.4.S Sequence alignment of LK-ADH with LB-ADH. The typical coenzyme binding site, GXXXGXG, of short-chain ADHs is shaded in gray.

Substrate binding also induces a conformational change in this enzyme. When both coenzymes and substrate bind the "closed" conformation of the enzyme is formed by a rotation of the catalytic domains of the two subunits relative to the coenzyme-binding do-mains.50 51a Structures of ternary complexes with inhibitors and with substrates have also been established. 

A crystal structure of a ternary complex of horse liver alcohol dehydrogenase with NADH and the inhibitor, dimethyl sulfoxide, first at 4.5 A resolution1365 and a further refinement to 2.9 A resolution,1366 has been published by Eklund et al. The gross structure of the ternary complex is similar to that of the free enzyme structure. Each subunit is divided into a coenzyme-binding domain and a catalytic domain. The subunits are joined together near the... 

The area of coenzyme binding within the enzyme is well established 1343 about 140 amino acid residues build up a pattern of pleated sheets surrounded by ar-helices which leads to the creation of a specific crevice for binding of the coenzyme via the adenine. 

Some early kinetic studies on the enzymic reaction indicated that LADH exhibits pre-steady state half-of-the-sites reactivity. Bernard et al. reported that two distinct kinetic processes, well separated in rate, were observed for the conversion of reactants into products under conditions of excess enzyme.1367 They also reported that each of the two phases corresponded to conversion of exactly one half of the limiting concentration of substrate being converted to products. On the basis of this they proposed two possible models, the favoured one based on catalytically non-equivalent but interconvertible states of the two binding sites, with the possibility that the asymmetry of the sites may be induced by coenzyme binding. Further evidence for this non-equivalence of the subunits was obtained in similar subsequent studies using a chromophoric nitroso substrate, p-nitroso-A,JV-dimethylaniline with limiting NADH concentrations.1368... 

However, in their study of intermediates in the enzymic reduction of acetaldehyde, Shore and Gutfreund could find no inequivalence in the binding sites of the subunits at all NADH concentrations studied.1369 This conclusion that the two active sites are kinetically equivalent is supported by kinetic studies by Hadom et al.1370 and by Kvassman and Pettersson. 1 Work by Kordal and Parsons also supports this conclusion.13" They devised a method of persuading 3H-labelled NADH to bind to one site per enzyme molecule and then, using a stopped-flow technique, to react this with excess unlabelled product. Full site reactivity was observed in either direction. They concluded that no half site reactivity was observed, and that there was no indication of subunit asymmetry induced by either the coenzyme binding or by chemical reaction. 

Carboxymethylation. It was found by Vallee and Li that one cysteine residue per subunit may be selectively carboxymethylated with iodoacetate.1405 Since this reaction causes deactivation of the enzyme, this cysteine residue, later identified as Cys-46,1406 was suggested to be at the active site. The deactivated carboxymethylated enzyme still binds NAD+. The carboxymethylation of this residue is preceded by a reversible binding of iodoacetate to the enzyme.1407 This observation has helped to identify an anion-binding site in the coenzymebinding domain, where the pyrophosphate group of the coenzyme binds. 

Ordered mechanisms often occur in the reactions of the NAD+-linked dehydrogenases, with the coenzyme binding first. The molecular explanation for this is that the binding of the dinucleotide causes a conformational change that increases the affinity of the enzyme for the other substrate (see Chapter 16). 

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