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

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• 作者：Vaibhav Pandya ; Mary Krishna Ekka ; Rajesh Kumar Dutta ; S. Kumaran  ; ;   ; ^{skumaran@imtech.res.in}
• 关键词：Mass spectrometry ; Enzyme kinetics ; Peptidases ; Carnosine ; Dipeptides
• 刊名：Analytical Biochemistry
• 出版年：2011
• 出版时间：1 November 2011
• 年：2011
• 卷：418
• 期：1
• 页码：134-142
• 全文大小：506 K

文摘

Chemical modifications of substrate peptides are often necessary to monitor the hydrolysis of small bioactive peptides. We developed an electrospray ionization mass spectrometry (ESI¨CMS) assay for studying substrate distributions in reaction mixtures and determined steady-state kinetic parameters, the Michaelis¨CMenten 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 19.5 s^?, 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 (73.8 s^?). 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¨CMS method are consistent with results of a reverse-phase high-performance liquid chromatography (RP¨CHPLC)-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¨CMS 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.