title: Sorbitol dehydrogenase from bovine lens: purification and properties.
abstract: Bovine lens sorbitol dehydrogenase (L-iditol:NAD+ 2-oxidoreductase, EC 1.1.1.14) (SDH) was purified to electrophoretic homogeneity (51 U/mg of protein) and characterized for both kinetic and some structural properties. The enzyme proves to be a homotetramer of 156 kDa containing one equivalent of zinc ion per subunit. Metal chelators such as EDTA and 1,10-phenanthroline determine a loss of enzyme activity which can be specifically recovered by addition of either zinc or manganese ions. Inactivation induced not only by metal chelators but also by thiol reagents is effectively prevented by the pyridine cofactor. Bovine lens SDH is active on polyalcohols and keto-sugars with more than three carbon atoms, and also requires special steric constraints for substrate recognition. Of the polyols, xylitol is the most effective substrate (kcat/KM of 8.1 s-1 mM-1), followed by sorbitol (kcat/KM of 1.59 s-1 mM-1); fructose, the most effective carbonyl substrate, displays a kcat/KM of only 0.9 s-1 mM-1. Analysis at the steady state of initial velocities as a function of the concentration of different substrates and cofactors and studies of product inhibition indicate for both fructose reduction and sorbitol oxidation a Theorell and Chance-type kinetic mechanism of action.
authors.affiliation: Dipartimento di Fisiologia e Biochimica, Università di Pisa, Italy.
authors.forename: I
authors.initials: I
authors.lastname: Marini
authors.forename: L
authors.initials: L
authors.lastname: Bucchioni
authors.forename: P
authors.initials: P
authors.lastname: Borella
authors.forename: A
authors.initials: A
authors.lastname: Del Corso
authors.forename: U
authors.initials: U
authors.lastname: Mura
mesh_terms: D000818:Animals; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D007064:L-Iditol 2-Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D007908:Lens, Crystalline / Q000201:enzymology*; D046911:Macromolecular Substances; D009243:NAD / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D009243:NAD; D007064:L-Iditol 2-Dehydrogenase