Solvent isotope partitioning: a new kinetic tool for the determination of desorption rates of reactant water from enzyme-substrate complexes in proteases.
The rates of desorption of the substrate water from the binary enzyme-H2O and ternary enzyme-H2O-(peptide)substrate complexes for the two hydrolases, porcine pepsin and thermolysin, have been investigated using a novel technique, solvent isotope partitioning. The experimental design of this method was based on the protocol of Rose et al. [Rose, I. A., O'Connell, E. L., Litwin, S., & BarTana, J. (1974) J. Biol. Chem. 249, 5163-5168] wherein the binary enzyme-H2(18)O complex established in the "pulse" solution was diluted into a "chase" solution containing variable concentrations of peptide substrates in a large pool of H2(16)O. The extent of trapping of H2(18)O within the respective E-H2(18)O and E-H2(18)O-(peptide)substrate complexes was determined from mass spectrometric analysis of the hydrolytic products. Our data have shown that the substrate water molecule of pepsin is not exclusively retained in the catalytic cycle and it desorbs from the apo- and substrate-bound complexes at rates that are at least 10 and 4 times faster, respectively, than that of product formation. Similarly, the low trapping of H2(18)O in the carboxylic product of the thermolysin reaction is a consequence of the ready desorption of H2(18)O from the ternary E-H2(18)O-(peptide)substrate complex and the binary E-H2(18)O complex. We attribute these results to the loss of the reactant water molecule due to desolvation of the enzyme's active site upon substrate binding.
Angeles
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Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
Roberts
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Carr
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Meek
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010103:Oxygen Isotopes; D010434:Pepsin A / Q000378:metabolism*; D012997:Solvents; D013552:Swine; D013820:Thermolysin / Q000378:metabolism*; D014867:Water / Q000378:metabolism*
D009842:Oligopeptides; D010103:Oxygen Isotopes; D012997:Solvents; D014867:Water; D010434:Pepsin A; D013820:Thermolysin