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Mass spectrometry assay for studying kinetic properties of dipeptidases: characterization of human and yeast dipeptidases.

DIR@IMTECH: CSIR-Institute of Microbial Technology

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Title Mass spectrometry assay for studying kinetic properties of dipeptidases: characterization of human and yeast dipeptidases.
 
Creator Pandya, Vaibhav
Ekka, Mary Krishna
Dutta, Rajesh Kumar
Kumaran, Sangaralingam
 
Subject QD Chemistry
 
Description Chemical modifications of substrate peptides are often necessary to monitor the hydrolysis of small bioactive peptides. We developed an electrospray ionization mass spectrometry (ESI-MS) assay for studying substrate distributions in reaction mixtures and determined steady-state kinetic parameters, the Michaelis-Menten constant (K(m)), and catalytic turnover rate (V(max)/[E](t)) for three metallodipeptidases: two carnosinases (CN1 and CN2) from human and Dug1p from yeast. The turnover rate (V(max)/[E](t)) of CN1 and CN2 determined at pH 8.0 (112.3 and 19.5s(-1), respectively) suggested that CN1 is approximately 6-fold more efficient. The turnover rate of Dug1p for Cys-Gly dipeptide at pH 8.0 was found to be slightly lower (73.8s(-1)). In addition, we determined kinetic parameters of CN2 at pH 9.2 and found that the turnover rate was increased by 4-fold with no significant change in the K(m). Kinetic parameters obtained by the ESI-MS method are consistent with results of a reverse-phase high-performance liquid chromatography (RP-HPLC)-based assay. Furthermore, we used tandem MS (MS/MS) analyses to characterize carnosine and measured its levels in CHO cell lines in a time-dependent manner. The ESI-MS method developed here obviates the need for substrate modification and provides a less laborious, accurate, and rapid assay for studying kinetic properties of dipeptidases in vitro as well as in vivo.
 
Publisher Elsevier Science
 
Date 2011-11-01
 
Type Article
PeerReviewed
 
Relation http://www.sciencedirect.com/science/article/pii/S000326971100426X
http://crdd.osdd.net/open/458/
 
Identifier Pandya, Vaibhav and Ekka, Mary Krishna and Dutta, Rajesh Kumar and Kumaran, Sangaralingam (2011) Mass spectrometry assay for studying kinetic properties of dipeptidases: characterization of human and yeast dipeptidases. Analytical biochemistry, 418 (1). pp. 134-42. ISSN 1096-0309