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Slow Tight Binding Inhibition of Proteinase K by a Proteinaceous Inhibitor

Jui Pandhare,C. Dash,M. Rao,V. Deshpande

Published 2003 in Journal of Biological Chemistry

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 × 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 × 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 × 10–3 s–1). The rate of dissociation of EI* (k6) was slower (4.5 ± 0.5 × 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 × 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.

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