Journal
JOURNAL OF PROTEOME RESEARCH
Volume 5, Issue 7, Pages 1667-1673Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr060033z
Keywords
carboxyl oxygen exchange; O-18 incorporation; mass spectrometry; lysyl endopeptidase; trypsin; quantitative proteomics; comparative proteomics
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Funding
- NCRR NIH HHS [P20RR017699, P20 RR016471-055390, P20 RR017699, P20 RR016741, P20 RR017699-057296] Funding Source: Medline
- NEI NIH HHS [R03 EY014020, R03 EY014020-04] Funding Source: Medline
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The pH dependency of the carboxyl oxygen exchange reaction catalyzed by lysyl endopeptidase (LysC) and trypsin has been studied. The reaction was quantitatively monitored by measuring the incorporation of O-18 atom into the alpha-carboxyl group of N-alpha-acetyl-L-lysine from H-2 O-18 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. Steadystate 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 O-18 labeling reactions and are thus pertinent to improving the accuracy and reliability of quantitative proteomic experiments utilizing O-18 labeling.
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