Glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130 (C130) was irreversibly inhibited in a time-dependent manner by two substrate analogs bearing side chains of variable length, namely 7␤-bromoacetyl aminocephalosporanic acid (BA-7-ACA) and 7␤-3-bromopropionyl aminocephalosporanic acid (BP-7-ACA). The inhibition of the enzyme with BA-7-ACA was attributable to reaction with a single amino acid residue within the ␤-subunit proven by comparative matrix assisted laser

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... zation-time of flight mass spectrometry. Further mass spectrometric analysis demonstrated that the fourth tryptophan residue of the ␤-subunit, Trp-B4, was alkylated by BA-7-ACA. By 1 H-13 C HSQC spectroscopy of C130 labeled by BA-2-13 C-7-ACA, it was shown that tryptophan residue(s) in the enzyme was alkylated, forming a carbon-carbon bond. Replacing Trp-B4 with other amino acid residues caused increases in K m , decreases in k cat , and instability of enzyme activity. None of the mutant enzymes except W-B4Y could be affinity-alkylated, but all were competitively inhibited by BA-7-ACA. Kinetic studies revealed that both BA-7-ACA and BP-7-ACA could specifically alkylate Trp-B4 of C130 as well as Tyr-B4 of the mutant W-B4Y. Because these alkylations were energy-requiring under physiological conditions, it is likely that the affinity labeling reactions were catalyzed by the C130 enzyme itself. The Trp-B4 residue is located in the middle of a characteristic ␣␤␤␣ sandwich structure. Therefore, a large conformational alteration during inhibitor binding and transition state formation is likely and suggests that a major conformational change is induced by substrate binding during the course of catalysis. The compound 7-aminocephalosporanic acid (7-ACA), 1 a key * This work was supported by National High Technology Development Program of China Grant 863-103-13-02-01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The amino acid sequence of this protein can be accessed through NCBI Protein Database under NCBI accession number 11386616. The atomic coordinates and structure factors (code 1GHD) have been deposited in the Protein