JBC, Vol. 251, Issue 19, 6150-6153, Oct, 1976
Active site chlorination of D-amino acid oxidase by N-chloro-D-leucine
D. J. Porter and H. J. Bright
N-Chloro-D-leucine is an irreversible inhibitor or D-amino acid oxidase on
a time scale of seconds. Studies with N-[36C]chloro-D-leucine,
N-chloro-D-[1-14C]leucine and N-chloro-D-[4,5-3H]leucine show that the
modified enzyme has been chlorinated at a site, or sites, on the apoenzyme.
The 36Cl measurements agree with titrations of catalytic activity in
showing that two chlorine equivalents are incorporated per active site
flavin. Kinetically, the interaction with N-chloro-D-leucine behaves in a
manner which is consistent with consecutive chlorinations of an amino acid
residue, or residues, in the active site region by the first 2 molecules of
N-chloro-D-leucine to be processed by the enzyme. The effect of
chlorination of the enzyme on the steady state parameters for oxidation of
D-alanine is entirely explained by a single perturbation, namely, a
1000-fold reduction in the specific rate of flavin reduction as measured
directly by rapid reaction techniques.
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