SUBSTRATE-SPECIFICITY OF SHEEP LIVER SORBITOL DEHYDROGENASE

The substrate specificity of sheep liver sorbitol dehydrogenase has be en studied by steady-state kinetics over the range pH 7-10. Sorbitol d ehydrogenase stereo-selectively catalyses the reversible NAD-linked ox idation of various polyols and other secondary alcohols into their cor responding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-alt ritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (k(cat)) and the substrate specificity-constants (k(cat)/K-m) are positively correlated with incr easing pH. The enzyme-catalysed reactions occur by a compulsory ordere d kinetic mechanism with the coenzyme as the first, or leading, substr ate. With many substrates, the rate-limiting step for the overall reac tion is the enzyme-NADH product dissociation. However, with several su bstrates there is a transition to a mechanism with partial rate-limita tion at the ternary complex level, especially at low pH. The kinetic d ata enable the elucidation of new empirical rules for the substrate sp ecificity of sorbitol dehydrogenase. The specificity-constants for pol yol oxidation vary as a function of substrate configuration with D-xyl o > D-ribo > L-xylo > D-lyxo approximate to L-arabino > D-arabino > L- lyxo. Catalytic activity with a polyol or an aromatic substrate and va rious l-deoxy derivatives thereof varies with -CH2OH>-CH2NH2> -CH2OCH3 approximate to -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is a lso nonessential for productive ternary complex formation and catalysi s. A predominantly nonpolar enzymic epitope appears to constitute an i mportant structural determinant for the substrate specificity of sorbi tol dehydrogenase. The existence of two distinct substrate binding reg ions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in so me polyols.