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The relations between the kinetic parameters for both sorbitol oxidation and fructose reduction by sheep liver sorbitol dehydrogenase show that a Theorell-Chance compulsory order mechanism operates from pH 7.4 to 9.9. This is supported by many parallels with the kinetics of horse liver alcohol dehydrogenase, which operates by this classical mechanism.An isotope-exchange study using U-('H8)sorbitol confirmed the existence of ternary complexes and that, under maximum velocity conditions, their interconversion is not rate-determining. Substrate inhibition at high concentrations of o-sorbitol or D-fructose confirmed rate-determining enzymecoenzyme product dissociation, slowed by the existence of more stable abortive ternary enzymccoenzyme product complexes with substrate. The effect of the inhibitor/activator 2,2,2-tribromoethanol showed the existence of enzyme-NAD-CBr3CH20H complexes inhibiting the first phase of reaction and enzyme-NADH-CBr3CH20H complexes dissociating more rapidly than the usual ratedetermining enzyme-NADH coenzyme product dissociation in the final phase. Inhibition studies with dithiothreitol also confirmed an ordered binding of coenzymes and second substrates to sorbitol dehydrogenase. Neither o-sorbitol nor D-fructose had any effect on enzyme inactivation by the affinity labelling reagent n~-2-bromo-3-(5-imidazolyl)propionic acid, thus giving no evidence for their existence as binary enzyme-substrate complexes.Several alternative polyol substrates for sorbitol dehydrogenase gave the same maximum velocity as sorbitol. This indicated a common rate-limiting binary enzyme-NADH product dissociation and a similarity of mechanism.An enzyme assay for pH 7.0 and 9.9 is given which enables the concentration of sorbitol dehydrogenase to be determined from initial rate measurements of enzyme activity.Sorbitol dehydrogenase (SDH) and aldose reductase constitute the sorbitol or polyol pathway, which functions as an important bypass to glycolysis and the pentose phosphate pathway in the metabolism of glucose, via u-sorbitol to Dfructose [l, 21. Operation of the sorbitol pathway has been implicated in the accumulation of sorbitol in the lens, leading to diabetic cataractogenesis [3], as well as other diabetic complications [4]. Because of the sorbitol pathway, the elucidation of the kinetic mechanism of action of sorbitol dehydrogenase is of particular interest.Sorbitol dehydrogenase catalyzes the reversible reaction

Iodoacetate alkylates and irreversibly inhibits horse-liver alcohol dehydrogenase. A reversible 1. Rate of inactivation is not proportional to iodoacetate concentration, but, rather, follows 2. The enzyme is protected competitively by other fatty-acids, and also by chloride. 3. The dissociation-constants of iodoacetate, and formate, and chloride all vary similarly with pH (increasing a t high pH, and suggesting a pK = 8.9-9.1).These results are discussed in terms of a model, and it is suggested that the reversible enzymeiodoacetate complex may be stable, rather than a necessary intermediate for alkylation of the enzyme.The great similarity of the results to those of another zinc-enzyme, carbonic anhydrase, is discussed; and it is suggested that anions bind to a zinc atom in the enzyme.enzyme-iodoacetate complex also forms, as deduced from the following evidence :Michaelis-Menten-type kinetics.Alcohol dehydrogenase from horse-liver has two cysteine groups per molecule which are reactive towards iodoacetate [1,2]. Evans and Rabin [2] used this to study the binding of several ligands to the enzyme. They reported that the rate of loss of enzyme activity was directly proportional to the concentration of iodoacetate : however, the experiments reported below show a saturation effect, and MichaelisMenten-type kinetics apply. Moreover, the enzyme is competitively protected by fatty acids, and by chloride. This enables the dissociation constants of complexes between enzyme and these anions to be calculated. Compounds which form binary complexes also tend to form ternary complexes with the enzyme and one or both forms of the coenzyme [3,4]. Therefore a simple independent method for measuring the formation of binary complexes may prove very useful. MATERIALS AND METHODSHorse-liver alcohol dehydrogenase was purchased as a crystalline suspension from C. F. Boehringer & Soehne (Mannheim) : before use, samples were centrifuged, the enzyme was dissolved, and dialysed against three or four changes of phosphate buffer (ionic strength 0.1, pH 7.4) for 3 days a t 4". The enzyme was centrifuged, and assayed as described previously Enzymes. Alcohol dehydrogenase or alcohol : NAD+ oxidoreductase (EC 1.1.1.1); carbonic anhydrase or carbonate hydro-lyase (EC 4.2.1.1). [5,6]. Assuming an extinction coefficient ( E f F$ml) a t 280 nm of 0.45, the apparent purity was 87-93 Olio.Iodoacetic acid was purchased from Sigma Chemical Co. (St. Louis, Mo.) and recrystallised twice from petroleum ether. It was white, with meltingpoint 81.1-81.7": total iodine content was 68.26O/,. Before use it was neutralised to pH 4.7-5.3 with sodium hydroxide. Iodoacetamide was purchased from Sigma Chemical Co. and recrystallised from ethanol-water and then water before use. It was white with melting-point 92.2-92.4" and total-iodine content 68.300/,. NAD+ was purchased from P-L Biochemicals (Milwaukee, Wis.) and used without further purification. Other chemicals were of reagent grade. Distilled water was redistilled in quartz apparatus.Enzyme inactivations were c...

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Purified Drosophila lebanonensis alcohol dehydrogenase (Adh) revealed one enzymically active zone in starch gel electrophoresis at pH 8.5. This zone was located on the cathode side of the origin. Incubation of D. lebanonensis Adh with NAD+ and acetone altered the electrophoretic pattern to more anodal migrating zones. D. lebanonensis Adh has an Mr of 56,000, a subunit of Mr of 28 000 and is a dimer with two active sites per enzyme molecule. This agrees with a polypeptide chain of 247 residues. Metal analysis by plasma emission spectroscopy indicated that this insect alcohol dehydrogenase is not a metalloenzyme. In studies of the substrate specificity and stereospecificity, D. lebanonensis Adh was more active with secondary than with primary alcohols. Both alkyl groups in the secondary alcohols interacted hydrophobically with the alcohol binding region of the active site. The catalytic centre activity for propan-2-ol was 7.4 s-1 and the maximum velocity of most secondary alcohols was approximately the same and indicative of rate-limiting enzyme-coenzyme dissociation. For primary alcohols the maximum velocity varied and was much lower than for secondary alcohols. The catalytic centre activity for ethanol was 2.4 s-1. With [2H6]ethanol a primary kinetic 2H isotope effect of 2.8 indicated that the interconversion of the ternary complexes was rate-limiting. Pyrazole was an ethanol-competitive inhibitor of the enzyme. The difference spectra of the enzyme-NAD+-pyrazole complex gave an absorption peak at 305 nm with epsilon 305 14.5 X 10(3) M-1 X cm-1. Concentrations and amounts of active enzyme can thus be determined. A kinetic rate assay to determine the concentration of enzyme active sites is also presented. This has been developed from active site concentrations established by titration at 305 nm of the enzyme and pyrazole with NAD+. In contrast with the amino acid composition, which indicated that D. lebanonensis Adh and the D. melanogaster alleloenzymes were not closely related, the enzymological studies showed that their active sites were similar although differing markedly from those of zinc alcohol dehydrogenases.