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-ethylnmleimide. Modification of one residue leads to loss of 40-60x of the enzyme activity in both the forward (NAD' + NADPH -+ NADH + NADP') and reverse reaction. The strong inhibitory action of phosphate ions on the reverse reaction Eur. J . Biochem. 107, 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 lo5 M-' at 25 "C in 50 mM sodium phosphate pH 7.50. This reversibility indicates formation of a cyclic dithiolarsinite derivative with considerable ring strain. The activity ofp-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.Pyridine nucleotide transhydrogenase is potentially a very important enzyme in the bacterium Azotohucter vinelandii since it catalyzes the transfer of reduction equivalents from the NADP pool to the NAD pool or vice versa. Since the equilibrium constant of the reaction NADPH + NAD' +NADH + NADP' is approximately equal to unity [I], it follows that the enzyme will cease to catalyze a net transfer when the degrees of reduction of the two pools are approximately equal. Since a lot of NADPH is generated in the catabolism of A . vinelundii [2], the steady-state degree of reduction of the NADP pool is likely to be higher than that of the NAD pool. This is certainly the case in systems containing a membrane-bound, energy-linked transhydrogenase, e.g. mitochondria and Escherichia coli [3,4]. The normal direction of transfer of reduction equivalents in A . vinelandii catalyzed by transhydrogenase is thus likely to be from the ~ Abbrrviulions. sNAD', thionicotinamide adenine dinucleotide, oxidized form; NADPH + sNAD+, shorthand notation for the reaction NADPH + sNAD+ + NADP' + sNADH; CIHgBzOH, p-chloromercuribenzoate; PhHgC1, phenylmercurichloride; HzNPhHgOAc, p-aminophenylmercuriacetate; Nbsz, 5:5'-dithiobis(2-nitrobenzoic acid); H2NPh-AsO, p-aminophenylar...

Cloning of thesthgene fromAzotobacter vinelandiiand construction of chimeric soluble pyridine nucleotide transhydrogenases

“…The amino acid composition of the STH as deduced from the sequence was found to be very similar to that determined by hydrolysis of the purified protein by Voordouw et al and Middelditch et al [11,12]. Titration experiments had shown that the enzyme contained three thiol residues, one of which was exposed to solvent [13], however, four cysteine residues are found in the sequence.…”

Cloning of the sth gene from Azotobacter vinelandii and construction of chimeric soluble pyridine nucleotide transhydrogenases

“…The amino acid composition of the STH as deduced from the sequence was found to be very similar to that determined by hydrolysis of the puri¢ed protein by Voordouw et al and Middelditch et al [11,12]. Titration experiments had shown that the enzyme contained three thiol residues, one of which was exposed to solvent [13], however, four cysteine residues are found in the sequence.…”

Structure and dynamics of a lipoic acid-arsenical adduct

“…The carbonyl carbon atom is not given, but it was found to resonate at 171.6 ppm. 6 Only determined for protonated carbon atoms. NOE's are given in parentheses.…”