Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase. The effect of pH on the aldehyde binding reactions and a re-examination of the problem of the site of proton release in the mechanism

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Abstract
Initial-rate measurements and stopped-flow spectrophotometric experiments over a wide range of pH implicate an enzyme group of pKa .apprx. 6.6 affecting the aldehyde binding reactions. It is possible, though not proved, that the group involved is the cysteine residue involved in catalysis. Stopped-flow fluorescence studies show that a group of pKa > 8.5 facilitates hydrolysis of the NADH-containing acyl-enzyme species. The identity of this group is quite unknown. Studies with 4-nitrobenzaldehyde that this substrate gives marked substrate inhibition at quite low (< 20 .mu.M) concentrations. The mechanism of catalysis seems to be the same as for propionaldehyde oxidation. It is argued that proton release occurs with both substrates on hydrolysis of the NADH-containing acyl-enzyme and not before hydride transfer, as has been previously suggested [Bennet, Buckley and Blackwell (1982) Biochemistry 21, 4407-4413].

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