title: 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 alpha-carboxyl group of N(alpha)-acetyl-L-lysine from H2(18)O 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(alpha)-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 Km (2-fold lower for Lys-C, 3-fold lower for trypsin), and higher kcat 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203, USA.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Hajkova
authors.forename: K C Sekhar
authors.initials: KC
authors.lastname: Rao
authors.forename: Masaru
authors.initials: M
authors.lastname: Miyagi
mesh_terms: D002245:Carbon Dioxide / Q000737:chemistry*; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010100:Oxygen / Q000378:metabolism*; D010103:Oxygen Isotopes / Q000737:chemistry; D040901:Proteomics / Q000379:methods; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism*; D014867:Water / Q000737:chemistry
chemical_list: D010103:Oxygen Isotopes; D014867:Water; D002245:Carbon Dioxide; C041069:carboxyl radical; D012697:Serine Endopeptidases; D014357:Trypsin; C036813:lysyl endopeptidase; D010100:Oxygen