Modification of the thiol residues of pyridine nucleotide transhydrogenase from Azotobacter vinelandii. Activity modulation by the divalent thiol reagent p-aminophenylarsenoxide.

Abstract

1. Purified pyridine nucleotide transhydrogenase from Azotobacter vinelandii contains three thiol residues as judged by titration with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions. 2. In the native conformation of the transhydrogenase only a single thiol residue is titrated. Modification of this exposed thiol does not influence transhydrogenase activity. 3. The two less exposed thiol residues can be reacted in part with either p-chloromercuribenzoate or N-ethyl-maleimide. Modification of one residue leads to loss of 40-60% of the enzyme activity in both the forward (NAD+ + NADPH leads to NADH + NADP+) and reverse reaction. The strong inhibitory action of phosphate ions on the reverse reaction [Voordouw et al. (1980) Eur. J. Biochem. 107, 337-344] is abolished after treatment with p-chloromercuribenzoate. Reaction with phenylmercurichloride or p-aminophenylmercuriacetate causes a similar activity loss without affecting the inhibitory action of phosphate. 4. The interaction of the divalent thiol inhibitor p-aminophenylarsenoxide with transhydrogenase was found to be reversible and is characterized by an association constant of 6.3 x 10(5) M-1 at 25 degrees C in 50 mM sodium phosphate pH 7.50. This reversibility indicates formation of a cyclic dithiolarsinite derivative with considerable ring strain. The activity of p-aminophenylarsenoxide-transhydrogenase is modulated by phosphate and magnesium ions. The activity of the transhydrogenase . p-aminophenylarsenoxide complex in the forward reaction is inhibited by phosphate and stimulated by magnesium ions. The reverse reaction is not catalyzed by the enzyme-inhibitor complex. 5. The presence of an activity modulating site in transhydrogenase which binds phosphate ions and has the two less exposed thiol residues in close proximity is indicated by the results.