Evidence for the importance of cysteine and arginine residues in Pseudomonas fluorescens UK-1 pantoate dehydrogenase.

Homogeneous pantoate dehydrogenase (D-pantoate:NAD+ 4-oxidoreductase, EC 1.1.1.106) was shown to be sensitive to inactivation by p-chloromercuribenzoate (100 microM), 5,5'-dithiobis(2-nitrobenzoic acid) (1 mM), iodoacetic acid (1 mM) and phenylglyoxal (5.3 mM). Potassium D-pantoate and NAD protected against inactivation by p-chloromercuribenzoate, 5,5'-dithiobis (2-nitrobenzoic acid) and iodoacetic acid. NAD and D-pantoate also provided substantial protection against inactivatrion by phenylglyoxal. Titration of the sulphydryl groups by 5,5'-dithiobis(2-nitrobenzoic acid) and incorporation of [14C]carboxymethyl revealed that there are two cysteine residues which are modified and one of those is essential for activity. In the presence of NAD and D-pantoate, incorporation of [14C]phenylglyoxal was decreased by 0.42 mol/mol of subunit.