Abstract：
:Pepstatin is a slow and tight-binding inhibitor of pepsin. Preincubating enzyme and inhibitor in H2O and in D2O in the absence of substrate generates an inverse solvent isotope effect of Dk = 0.69 +/- 0.06 on the apparent first-order rate constant for the decay in enzymatic activity. Proton inventory analysis of the inverse isotope effect suggests a single transition-state proton with a fractionation factor of 1.41 +/- 0.05. In contrast, combining enzyme with inhibitor and substrate (Leu-Ser-p-nitro-Phe-Nle-Ala-Leu-OMe) simultaneously along with observing the decay in enzymatic activity during catalytic turnovers generates a normal solvent isotope effect of Dk = 1.25 +/- 0.09. Proton inventory analysis of the normal isotope effect suggests a single reactant-state proton with a fractionation factor of 1.46 +/- 0.03. These two experimental designs are often considered equivalent, but the differences in isotopic data require that the pathway for onset of pepstatin inhibition in the absence of substrate must be different from the pathway in the presence of substrate. In the former, the inhibitor can only bind to free enzyme; in the latter, the inhibitor is hindered from binding to free enzyme because of competition with substrate but can bind to intermediate forms of enzyme generated during catalytic turnovers, downstream from enzyme-product complexes.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Cho YK,Rebholz KL,Northrop DBdoi
10.1021/bi00198a032subject
Has Abstractpub_date
1994-08-16 00:00:00pages
9637-42issue
32eissn
0006-2960issn
1520-4995journal_volume
33pub_type
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