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 alpha-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, Asp(10), 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 Asn(10) attacks the substrate to form an imidate, and a proton and d-lactic acid are eliminated to produce a nitrile (beta-cyanoalanine residue), followed by hydrolysis to reproduce Asn(10). This is the first report of the function of Asn to catalyze nucleophilic substitution through its conversion to beta-cyanoalanine residue as an intermediate structure. Also, these results demonstrate that mass spectrometry is remarkably useful in monitoring enzyme reactions.
Ichiyama
S
S
Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Kurihara
T
T
Li
Y F
YF
Kogure
Y
Y
Tsunasawa
S
S
Esaki
N
N
D000409:Alanine / Q000378:metabolism*; D001216:Asparagine; D002384:Catalysis; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D011549:Pseudomonas / Q000201:enzymology*
D001216:Asparagine; D006867:Hydrolases; C034507:2-haloacid dehalogenase; D000409:Alanine