Novel catalytic mechanism of nucleophilic substitution by asparagine residue involving cyanoalanine intermediate revealed by mass spectrometric monitoring of an enzyme reaction

L-2-Haloacid dehalogenase from Pseudomonas sp. YL catalyzes the hydrolytic dehalogenation, in which Asp(10) acts as a nucleophile to attack the Lu-car bon of L-2-haloalkanoates to form an ester intermediate, which is subsequen tly: hydrolyzed to produce D-2-hydroxyalkanoates. Surprisingly, replacement of the catalytic residue, Asp(10), by Asn did not result in total inactiva tion of the enzyme (Kurihara, T., Liu, J.-Q., Nardi-Dei, V., Koshikawa, H., Esaki, N., and Soda, K. (1995) J. Biochem. 117, 1917-1322). 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 in cubated with the substrate, L-2-chloropropionate. LC/MS and tandem MS/MS an alyses revealed that Asn(10) attacks the substrate to form an imidate, and a proton and D-lactic acid are eliminated to produce a nitrile (beta -cyano alanine residue), followed by hydrolysis to reproduce Asn(10). This is the first report of the function of Asn to catalyze nucleophilic substitution t hrough its conversion to beta -cyanoalanine residue as an intermediate stru cture. Also, these results demonstrate that mass spectrometry is remarkably useful in monitoring enzyme reactions.