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 [14C]PGO revealed that the arginyl residue was in the beta subunit of the enzyme. The fragment containing the 14C-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.
Mukouyama
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Department of Biosciences, Faculty of Science, Kitasato University, Sagamihara, Kanagawa, 228-8555, Japan. mukoyama@jet.sci,kitasato-u.ac.jp
Hirose
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T
Suzuki
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D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001120:Arginine; D017124:Conserved Sequence; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D010658:Phenylglyoxal / Q000737:chemistry* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D016415:Sequence Alignment
D001120:Arginine; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase; D010658:Phenylglyoxal