pH Dependency of the Carboxyl Oxygen Exchange Reaction Catalyzed by Lysyl Endopeptidase and Trypsin

Abstract

The pH dependency of the carboxyl oxygen exchange reaction catalyzed by lysyl endopeptidase (Lys-C) and trypsin has been studied. The reaction was quantitatively monitored by measuring the incorporation of ¹⁸O atom into the α-carboxyl group of N^α-acetyl-l-lysine from H₂¹⁸O solvent. The optimum pHs of the carboxyl oxygen exchange reaction catalyzed by Lys-C and trypsin were found to be pH 5.0 and 6.0, respectively, which were significantly shifted toward acidic pHs compared to the most favorable pHs of their amidase activities for N^α-acetyl-l-lysine amide in the pHs examined. Steady-state kinetics parameters were also determined for both enzymes at two different pHs, one at the pH optimum for their carboxyl oxygen exchange activity (pH 5−6) and the other at the favorable pH for their amidase activity (pH 8−9). Significantly lower K_m (2-fold lower for Lys-C, 3-fold lower for trypsin), and higher k_cat values (1.5-fold higher for Lys-C, 5-fold higher for trypsin) were obtained at the acidic pHs compared to the alkaline pHs, suggesting that Lys-C and trypsin have higher substrate binding affinities and higher catalytic rates at the acidic pHs than at the alkaline pHs. The higher carboxyl oxygen exchange activities at the acidic pHs were also confirmed with peptide substrates derived from apomyoglobin. These findings are significant toward the goal of improving the efficiency of the Lys-C and trypsin catalyzed ¹⁸O labeling reactions and are thus pertinent to improving the accuracy and reliability of quantitative proteomic experiments utilizing ¹⁸O labeling. Keywords: carboxyl oxygen exchange • ¹⁸O incorporation • mass spectrometry • lysyl endopeptidase • trypsin • quantitative proteomics • comparative proteomics