TY  - JOUR
ID  - open458
UR  - http://www.sciencedirect.com/science/article/pii/S000326971100426X
IS  - 1
A1  - Pandya, Vaibhav
A1  - Ekka, Mary Krishna
A1  - Dutta, Rajesh Kumar
A1  - Kumaran, Sangaralingam
N2  - 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.
VL  - 418
TI  - Mass spectrometry assay for studying kinetic properties of dipeptidases: characterization of human and yeast dipeptidases.
AV  - none
EP  - 42
N1  - Copyright of this article belongs to Elsevier Science.
Y1  - 2011/11/01/
PB  - Elsevier Science
JF  - Analytical biochemistry
KW  - Mass spectrometry; Enzyme kinetics; Peptidases; Carnosine; Dipeptides
SN  - 1096-0309
SP  - 134
ER  -