Functions of aldehyde reductases.

dehydrogenases. The same effects obtained when e N A D + is bound to binary complexes confirm the transfer of the coenzyme to a more hydrophobic environment, but also indicate that the coenzyme is bound to ODH in an open conformation, as found in other dehydrogenases. The high degree of conformational rigidity of bound coenzyme were deduced on the one hand from the negligible temperature coefficient of the ellipticity band of ODH (Luisi et al., 1977) and, on the other hand, from the study of fluorescence-anisotropy decay of bound NADH (Brochon et al., 1977). A peculiar feature of ODH-coenzyme complexes is the temperature-independence of the dissociation constants (Luisi et al., 1975) over a temperature range in which the binding of the coenzyme to some other dehydrogenases changes by one order of magnitude. This appears to be due to a biologically relevant structure-function relationship of the enzyme active site (Luisi et al., 1977). To study essential amino acid residues we have used principally the method of chemical modification. ODH contains three accessible -SH groups, only one of which is essential for activity, and it seems to be situated near the adenine-binding site (Olomucki et al., 1972, 1975). Our attention was principally attracted by the imidazole group, which could play a crucial role in the oxidoreduction process. A histidine residue has been found in the catalytic centre of many dehydrogenases; this was confirmed by crystallographic data (Rossmann et al., 1977). By using initially diethyl pyrocarbonate as a histidine reagent (Huc et al.. 1971). and, in later experiments, dye-sensitized photo-oxidation (Thom6Beau et al., 1973), it has been shown that two histidine residues are accessible to chemical modification, but only one of them seems to be essential for activity. We have made preliminary attempts to modify covalently the essential histidine residue in order to isolate the peptide containing this residue. For this purpose Dr. D. B. Pho in our laboratory has used bromopyruvate (Berghauser et al., 1971) as an affinity-labelling reagent. Bromopyruvate itself is a poor substrate and is an active-site-directed inhibitor that seems to modify only histidine residue(s). The possibility of the existence of a charge relay system involving histidine and dicarboxylic amino acid residues in the active site of some dehydrogenases led us to examine whether a carboxy group was essential for ODH activity. We used a water-soluble carbodi-imide for the modification of these groups (Huc et al., 1975). The results of this treatment allowed us to conclude that the inhibition of ODH is accompanied by the modification of one carboxy group, which is situated near the substrate-binding site. We have recently prepared very promising crystals of ODH (Fig. 1.) and hope to perform a crystallographic study in order to characterize the catalytic centre and to confirm structural analogies of the coenzyme-binding domain between ODH and other dehydrogenases. Fig. 1. Octopine dehydrogenase crystals obtained by the hanging-droplet method