Formaldehyde dehydrogenase from Pseudomonas putida : The role of a cysteinyl residue in the enzyme activity.

Abstract

Formaldehyde dehydrogenase from Pseudomonas putida C-83 was found to contain 7 halfcystine residues per subunit monomer, as checked by the method of performic acid oxidation. Approximately 7 sulfhydryl groups per subunil monomer were titrated with 5, 5, -dithiobis(2-nitrobenzoic acid) (DTNB) after denaturation with 8 M urea. In the native enzyme, modification of three sulfhydryl groups per subunit with p-chloromercuribenzoate (PCMB) led to the complete loss of enzyme activities for both formaldehyde and n-butanol. Hydrogen-peroxide competitively inhibited the enzyme activity for formaldehyde, while it was only slightly inhibitory to the activity for n-butanol. Both formaldehyde and hydrogen-peroxide protected one sulfhydryl group per subunit monomer from modification with PCMB. Moreover, hydrogen-peroxide was hardly reactive to the enzyme which was preincubated with formaldehyde. From these observations, we conclude that one of three PCMB-reactive sulfhydryl groups is essential for the binding of formaldehyde, and hydrogen-peroxide modifies this sulfhydryl group.