Chemical modification of L-phenylalanine oxidase from Pseudomonas sp. P-501 by phenylglyoxal.: Identification of one essential arginyl residue

L-Phenylalanine oxidase from Pseudomonas sp. P-501 was irreversibly inactivated by the arginine-specific reagents, phenylglyoxal (PGO) and p-hydroxyphenylglyoxal (HPG), The inactivation by PGO and HPG follows pseudo-first-order kinetics with second-order rate constants of 10.6 and 15.1 M-1.min(-1), respectively, and a single arginyl residue was modified specifically. The effective protection by substrate L-phenylalanine against the inactivation by these reagents strongly suggests that the arginyl residue is located in the substrate binding site. SDS/PAGE analysis of the enzyme modified with [C-14] PGO revealed that the arginyl residue was in the beta subunit of the enzyme, The fragment containing the C-14-labeled arginyl residue was purified from the enzymatic digests of the labeled beta subunit by HPLC and sequenced. The modification of Arg-35 in the beta subunit was identified. The sequence around Arg-35 shows homology to the corresponding regions of tryptophan-2-monooxygenases.