Abstract
The kinetics of slow onset inhibition of Proteinase K by a proteinaceous alkaline protease inhibitor (API) from a Streptomyces sp. is presented. The kinetic analysis revealed competitive inhibition of Proteinase K by API with an IC50 value 5.5 +/- 0.5 x 10-5 m. The progress curves were time-dependent, consistent with a two-step slow tight binding inhibition. The first step involved a rapid equilibrium for formation of reversible enzyme-inhibitor complex (EI) with a Ki value 5.2 +/- 0.6 x 10-6 m. The EI complex isomerized to a stable complex (EI*) in the second step because of inhibitor-induced conformational changes, with a rate constant k5 (9.2 +/- 1 x 10-3 s-1). The rate of dissociation of EI* (k6) was slower (4.5 +/- 0.5 x 10-5 s-1) indicating the tight binding nature of the inhibitor. The overall inhibition constant Ki* for two-step inhibition of Proteinase K by API was 2.5 +/- 0.3 x 10-7 m. Time-dependent dissociation of EI* revealed that the complex failed to dissociate after a time point and formed a conformationally altered, irreversible complex EI**. These conformational states of enzyme-inhibitor complexes were characterized by fluorescence spectroscopy. Tryptophanyl fluorescence of Proteinase K was quenched as a function of API concentration without any shift in the emission maximum indicating a subtle conformational change in the enzyme, which is correlated to the isomerization of EI to EI*. Time-dependent shift in the emission maxima of EI* revealed the induction of gross conformational changes, which can be correlated to the irreversible conformationally locked EI** complex. API binds to the active site of the enzyme as demonstrated by the abolished fluorescence of 5-iodoacetamidofluorescein-labeled Proteinase K. The chemoaffinity labeling experiments lead us to hypothesize that the inactivation of Proteinase K is because of the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the catalytic triad and other residues involved in catalysis.
Highlights
The kinetics of slow onset inhibition of Proteinase K by a proteinaceous alkaline protease inhibitor (API) from a Streptomyces sp. is presented
Initial inhibition kinetic assessments revealed that API is a competitive inhibitor of Proteinase K with an IC50 value of 5.5 Ϯ 0.5 ϫ 10Ϫ5 M (Fig. 1)
We present the first report of a slow tight binding proteinaceous inhibitor (API) of the alkaline protease Proteinase K
Summary
Materials—Purified Proteinase K from T. album limber, 5-iodoacetamidofluorescein, and sAAPF-p-nitroanilide were obtained from Sigma. For the Lineweaver-Burk analysis, Proteinase K (1 M) was incubated with API at 1 and 2.5 M and assayed at increasing concentrations of casein (1–10 mg/ml). In the method of Dixon [18], proteolytic activity of Proteinase K (1 M) was measured in the presence of 5 and 10 mg/ml of casein, at concentrations of API ranging from 1 to 5 M at 37 °C for 30 min. Assays were carried out in a 1-ml reaction mixture containing enzyme, substrate, and inhibitor at various concentrations. The reaction mixture contained Proteinase K (100 nM) in the required buffer and varying concentrations of API and casein (5 mg/ml). The progress curves were analyzed by Equations 2 and 5 using non-linear least-square parameter minimization to determine the bestfit values with the corrections for the tight binding inhibition. Values for Ki were obtained from Dixon analysis at a constant substrate concentration as described in Equation 7
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