JBC, Vol. 251, Issue 20, 6179-6182, Oct, 1976
D-Serine dehydratase from Escherichia coli. Essential arginine residue at the pyridoxal 5'-phosphate binding site
M. N. Kazarinoff and E. E. Snell
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.
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