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

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 (Km), and catalytic turnover rate (Vmax/[E]t) for three metallodipeptidases: two carnosinases (CN1 and CN2) from human and Dug1p from yeast. The turnover rate (Vmax/[E]t) of CN1 and CN2 determined at pH 8.0 (112.3 and 19.5 s−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 (73.8 s−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 Km. 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.