Abstract
Initial-rate measurements and stopped-flow spectrophotometric experiments over a wide range of pH implicate an enzyme group of pKa approximately 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 greater than 8.5 facilitates hydrolysis of the NADH-containing acyl-enzyme species. The identity of this group is quite unknown. Studies with 4-nitrobenzaldehyde show that this substrate gives marked substrate inhibition at quite low (less than 20 microM) 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 [Bennett, Buckley & Blackwell (1982) Biochemistry 21, 4407-4413].
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