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J. Biol. Chem., Vol. 275, Issue 52, 40804-40809, December 29, 2000

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Novel Catalytic Mechanism of Nucleophilic Substitution by Asparagine Residue Involving Cyanoalanine Intermediate Revealed by Mass Spectrometric Monitoring of an Enzyme Reaction^*

Susumu Ichiyama, Tatsuo Kurihara, Yong-Fu Li, Yoshifumi Kogure^§, Susumu Tsunasawa^§, and Nobuyoshi Esaki^¶

From the  Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan and the ^§ Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Kusatsu, Shiga 525-0055, Japan

L-2-Haloacid dehalogenase from Pseudomonas sp. YL catalyzes the hydrolytic dehalogenation, in which Asp¹⁰ acts as a nucleophile to attack the -carbon of L-2-haloalkanoates to form an ester intermediate, which is subsequently hydrolyzed to produce D-2-hydroxyalkanoates. Surprisingly, replacement of the catalytic residue, Asp¹⁰, by Asn did not result in total inactivation of the enzyme (Kurihara, T., Liu, J.-Q., Nardi-Dei, V., Koshikawa, H., Esaki, N., and Soda, K. (1995) J. Biochem. 117, 1317-1322). In this study, we monitored the D10N mutant enzyme reaction by ion-spray mass spectrometry, and found that the enzyme shows a unique structural change when it was incubated with the substrate, L-2-chloropropionate. LC/MS and tandem MS/MS analyses revealed that Asn¹⁰ attacks the substrate to form an imidate, and a proton and D-lactic acid are eliminated to produce a nitrile (-cyanoalanine residue), followed by hydrolysis to reproduce Asn¹⁰. This is the first report of the function of Asn to catalyze nucleophilic substitution through its conversion to -cyanoalanine residue as an intermediate structure. Also, these results demonstrate that mass spectrometry is remarkably useful in monitoring enzyme reactions.

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