Chlorination of an active site tyrosyl residue in D-amino acid oxidase by N-chloro-D-leucine.

The chlorination of the flavoenzyme D-amino acid oxidase (EC 1.4.3.3, from hog kidney) by N-chloro-D-leucine, which specifically slows flavin reduction by a factor of 2 x 10(3), has been studied with respect to the site and mechanism of chlorination and the chlorinated enzyme has been extensively characterized. Our major results are as follows: 1. The oxidized holoenzyme (E0) incorporates 2 eq of 36Cl/flavin site from D-[N-36Cl]leucine. Evidence from amino acid and 36Cl analysis, isotopic dilution, and spectral titrations shows that the target residue is tyrosine and that this is converted to 3,5-dichlorotyrosine. The resulting decrease in basicity of this tyrosine (similar to 10(3)-fold) parallels the decrease (2 x 10(3)-fold) in the first order rate constant for flavin reduction observed with E0-Cl2 and we show that this residue satisfies all available criteria as being directly involved in the catalysis of flavin reduction. 2. Half-modified enzyme is a mixture of 0.5 E0 + 0.5 E0-Cl2 and E0-Cl is therefore chlorinated much more rapidly than E0 (kCl = 8 x 10(2) M-1S-1). Several lines of evidence show that enzyme-bound N-chloro-D-leucine is not oxidized prior to chlorination of the active site tyrosine. 3. The chlorination reaction is highly specific with respect to the structure of the chlorinating agent. Only the N-chloro derivatives of D-leucine, D-isoleucine, and D-norvaline are reactive while N-chloro derivatives of L-amino acids are tightly bound, but reversibly competitive, inhibitors which markedly perturb the electronic spectrum of enzyme-bound FAD.