title: Mass spectrometry assay for studying kinetic properties of dipeptidases: characterization of human and yeast dipeptidases.
abstract: 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.
authors.affiliation: Council of Scientific and Industrial Research, Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, India.
authors.forename: Vaibhav
authors.initials: V
authors.lastname: Pandya
authors.forename: Mary Krishna
authors.initials: MK
authors.lastname: Ekka
authors.forename: Rajesh Kumar
authors.initials: RK
authors.lastname: Dutta
authors.forename: S
authors.initials: S
authors.lastname: Kumaran
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004150:Dipeptidases / Q000737:chemistry* / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D004151:Dipeptides; D029701:Saccharomyces cerevisiae Proteins; C522928:CNDP1 protein, human; C523458:DUG1 protein, S cerevisiae; D004150:Dipeptidases