title: D-Serine dehydratase from Escherichia coli. Essential arginine residue at the pyridoxal 5'-phosphate binding site.
abstract: D-Serine apodehydratase from Escherichia coli is rapidly inactivated by butanedione in K+ borate buffer or by phenylglyoxal in K+ phosphate buffer at pH 8, 25 degrees. Pyridoxal-P protects against the inactivation. Modification of the apoenzyme abolishes its ability to bind the cofactor, pyridoxal-P, but the apparent Km for the substrate, D-serine, is not altered. The concentration dependence of the rate of butanedione inactivation in K+ borate buffer indicates that it is a two-step process with one butanedione bound per molecule of apoenzyme to give an inactive complex; half-maximal rate of inactivation is obtained at 37 mM butanedione. Butanedione inactivation is fully reversed following removal of excess reagent and borate. Similar studies with [14C]phenylglyoxal show that in the presence of pyridoxal-P at least 2 arginine residues may be modified without loss of activity. In the absence of pyridoxal-P modification of a single additional arginine residue results in loss of activity. Results with both inactivating reagents thus demonstrate that a critical arginine residue participates in binding of the coenzyme, pyridoxal-P. The stoichiometry of phenylglyoxal incorporation into the enzyme is different in the presence and absence of borate. Under both conditions incorporated phenylglyoxal is slowly lost on dialysis at neutral pH. A possible explanation of these effects is discussed.
mesh_terms: D001051:Apoenzymes / Q000378:metabolism; D001120:Arginine; D001665:Binding Sites; D003931:Diacetyl / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D012695:L-Serine Dehydratase / Q000378:metabolism*; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000494:pharmacology*
chemical_list: D001051:Apoenzymes; D011732:Pyridoxal Phosphate; D001120:Arginine; D012695:L-Serine Dehydratase; D003931:Diacetyl