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It is still a big challenge to describe how the conformation changes for an enzyme converting from ground state to transition state that may have life time near 10-13 sec,the time for a single bond vibration (13).A large scale conformational change around the non-solvent accessible Trpβ4 in cephalosporin acylase upon the ligand binding that was predicted by an affinity alkylation on 9.Here,we show two extraordinary enzymatic reactions in β-lactam acylases to probe the integer conformational dynamic.Firstly,we found that bromoacyl-7ACA interacts with penicillin acylase in two distinguish aspects,to be hydrolyzed as a substrate and to affinity alkylate the conserved Trpβ4 as an inhibitor after the substrate binding.