Determination of Km and Vmax for tryptic peptide hydrolysis using fast atom bombardment mass spectrometry

Abstract

23) Katakuse, I.; Nakabushi, H.; Ichihara, T.; Sakurai, T.; Matsuo, T.; Matsuda, H. Int. J . Mass Spectrom. Ion Proc. 1984, 62, 17. (24) Morgan, T. G.; RabrenoviE, M.; Harris, F. M.; Beynon, J. H. Org. Mass (32) Castro, M. E.; Mallis, L. M.; Russell, D H. J Am. Chem. SOC. 1985, 107, 5652. (33) Mallis, L. M.; Russell, D. H., unpublished results. (34) Castro, M. E.; Mallis, L. M.; Russell, D. H., unpublished results. (35) Castro, M. E.; Russell, D. H. Fast atom bombardment mass spectrometry was used to provide quantitative analytical data for the calculation of kinetic parameters for the action of trypsin on several small peptides. The method provides a unique capability in that spectfic molecular species may be followed independently of other components'in the mixture. The analysis takes place within minutes of sampling the reaction and does not require any cleanup or chemical derivatiratlon procedures prior to analysis. The method is illustrated by determining K, and k,, for the hydrolysis of several peptides by trypsin. These results are compared with values for similar peptides that have been reported in the literature.The determination of kinetic constants of enzymes for specific substrates has involved the use of a variety of analytical methods, which usually include spectroscopic and chromatographic techniques as primary quantitative tools. Much of this information has been obtained through the study of substrate analogues and chemically modified natural substrates in order to provide compounds that can be followed in the enzyme reaction with high sensitivity and specificity.Present address: Proctor a n d Gamble Corp., Cincinnati, OH.For example, kinetic constants for trypsin have been determined by using tosylarginine methyl ester and benzoylarginine ethyl ester, as well as various analogues. Although these data serve a valuable role in providing comparative kinetic constants for small substrates and the convenient assay of these enzymes, they do not necessarily represent constants that may be reasonably applied to estimate those for natural substrates.Work done thus far in the determination of K , for trypsin using specific peptide substrates has given values that are much greater than those for many synthetic substrates (1-3). For other enzymes, such data are sparse due to the difficulties encountered with the appropriate analytical methodology.Fast atom bombardment mass spectrometry (FABMS) is emerging as a powerful analytical tool for biomolecular analysis in studies involving the determination of ionic constituents in aqueous solutions (4). For enzyme kinetic studies, FABMS provides several basic capabilities. First, it has a very high degree of molecular specificity in that the mass spectrum can identify individual molecular species in mixtures, including specific charged forms of a given compound. Second, it can analyze most compounds directly in reaction mixtures without chemical derivatization and separation steps. Third, it is a very sensitive technique, which most often requires only a few nanomo...

Mass spectrometry for enzyme assays and inhibitor screening: An emerging application in pharmaceutical research

“…The resulting initial rates were comparable regardless of the method used, thus validating the mass spectrometry approach. Further studies using FAB‐MS to measure trypsin kinetics demonstrated K m and V max values for a number of peptide substrates that were consistent with literature values (Caprioli & Smith, 1986). These and numerous other early studies (review in Caprioli, 1987a,b), formed the groundwork for the use of mass spectrometry by biochemists and enzymologists to further understand their enzyme systems.…”

Label-free and real-time monitoring of trypsin activity in living cells by quantum-dot-based fluorescent sensors

“…4C, V max /K m was directly proportional to the concentration of trypsin, and the slope of this linear plot afforded a value for the specicity constant (k cat /K m , k cat is the catalytic constant or turnover number), k cat /K m ¼ 6150 M À1 s À1 , which was in good agreement with values obtained using other assays. 21,[35][36][37] These results demonstrated that it was possible to monitor trypsin in real time and calculate enzyme kinetic parameters on the basis of the uorescence change of the QDs-cyt.c complex.…”

Protein acidification and hydrolysis by pepsin ensure efficient trypsin-catalyzed hydrolysis

“…It is known that pepsin and trypsincatalysed hydrolysis, after different amino acid sequences, results in a wide range of kinetic parameters. 2,12,[44][45][46][47] It has been suggested that pepsin-catalysed hydrolysis of a peptide bond within a protein is more efficient than of short, synthetic peptides. 11 Our findings do not corroborate this.…”