Slow Tight Binding Inhibition of Proteinase K by a Proteinaceous Inhibitor
2003; Elsevier BV; Volume: 278; Issue: 49 Linguagem: Inglês
10.1074/jbc.m308976200
ISSN1083-351X
AutoresJui Pandhare, Chandravanu Dash, Mala Rao, Vasanti Deshpande,
Tópico(s)Biochemical and Molecular Research
ResumoThe 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 × 10–5m. 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 × 10–6m. 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 × 10–7m. 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. 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 × 10–5m. 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 × 10–6m. 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 × 10–7m. 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. An effervescence of research efforts has been expended in the design and synthesis of inhibitors of proteolytic enzymes not only to understand the active site structure and mechanism of these enzymes but also to help generate new therapeutic agents. Specific inhibitors of proteases have proved valuable in a number of applications ranging from mechanistic studies to possible therapeutic uses. Protein inhibitors of proteases are ubiquitously present in plants, animals, and microorganisms (1.Birk Y. Neuberger A. Brocklehurst K. Proteinase Inhibitors, Hydrolytic Enzymes. Elsevier Science Publishers B. V., Amsterdam1987: 257-305Google Scholar). Fundamentally, proteinaceous inhibitors should serve as substrates for proteolysis rather than being their inhibitors. Elucidation of this paradox is the basis for the extensive research on the structure-function relationship of proteinaceous inhibitors of proteases. The importance of proteolytic processes in the regulation of post-translational processing of precursor proteins and the involvement of proteases in intracellular protein metabolism and in various pathological processes have recently stimulated tremendous interest in studying the kinetic properties of naturally occurring target-oriented protease inhibitors. Serine proteases are divided in trypsin-like and subtilisin-like families, which have been independently evolved with a similar catalytic mechanism (2.Blow D.M. Acc. Chem. Res. 1976; 9: 142-145Crossref Scopus (482) Google Scholar, 3.Tsukuda H. Blow D. J. Mol. Biol. 1985; 184: 703-711Crossref PubMed Scopus (267) Google Scholar, 4.Bone R. Shenvi A.B. Kettner C. Agard D.A. Biochemistry. 1987; 26: 7609-7614Crossref PubMed Scopus (140) Google Scholar, 5.Siezen R.J. Willem M.V. Leunissen J.A.M. Dikstra B.W. Protein Eng. 1991; 4: 719-737Crossref PubMed Scopus (303) Google Scholar, 6.Lange G. Betzel C. Branner S. Wilson K.S. Eur. J. Biochem. 1994; 224: 507-518Crossref PubMed Scopus (23) Google Scholar). The functional importance of the catalytic triad and oxyanion hole in catalysis of serine proteases has been clearly established (7.Russel A.J. Fersht A.R. Nature. 1987; 328: 496-500Crossref PubMed Scopus (301) Google Scholar, 8.Carter P. Wells J.A. Nature. 1988; 332: 564-568Crossref PubMed Scopus (530) Google Scholar). Proteinase K, the serine protease from the subtilisin family is a highly active extracellular alkaline serine endopeptidase from Tritirachium album limber and named Proteinase K because of its ability to digest native keratin (9.Ebeling W. Hennrich N. Klockow M. Metz H. Orth H.U. Lang H. Eur. J. Biochem. 1974; 47: 91-97Crossref PubMed Scopus (483) Google Scholar, 10.Betzel C. Pal G.P. Saenger W. Eur. J. Biochem. 1988; 178: 155-171Crossref PubMed Scopus (159) Google Scholar). By virtue of its unusual stability at low concentrations of SDS and urea, Proteinase K has immense applications in basic and applied research. X-ray crystallographic studies have revealed that the catalytic triad of Proteinase K is formed by the residues His-69, Asp-39, Ser-224, and a single free Cys-73 residue is located below the functional His-69 (11.Betzel C. Gourinath S. Kumar P. Kaur P. Perbandt M. Eschenburg S. Singh T.P. Biochemistry. 2001; 40: 3080-3088Crossref PubMed Scopus (112) Google Scholar). During catalysis Ser-224 functions as the primary nucleophile and His-69 plays a dual role as proton acceptor and donor at different steps in the reaction. The Asp-39 residue is known to participate in a complex hydrogen bond network with His-69, thus bringing the His-69 residue in the correct orientation to facilitate nucleophilic attack by Ser-224. Determination of the mechanism of inhibition of this protease will provide better insights in understanding the mechanism of inhibition and will shed light on the protein-protein interactions at the molecular level. Serine proteases and their protein inhibitors have been the most intensively studied group of protein-protein complexes. An enormous number of known and partially characterized inhibitors of serine proteases from plants, animals, and microorganisms have been documented and have been grouped in different families (1.Birk Y. Neuberger A. Brocklehurst K. Proteinase Inhibitors, Hydrolytic Enzymes. Elsevier Science Publishers B. V., Amsterdam1987: 257-305Google Scholar, 12.Rao M. Tanksale A.M. Ghatge M.S. Deshpande V.V. Microbiol. Mol. Biol. Rev. 1998; 62: 597-635Crossref PubMed Google Scholar). Among the proteinaceous inhibitors from microorganisms, the well characterized inhibitors are from Streptomyces (13.Taguchi S. Kojima S. Kumagai I. Ogawara H. Miura K. Momose H. FEMS Microbiol. Lett. 1992; 99: 293-297Crossref Scopus (29) Google Scholar) and belong to the family Streptomyces subtilisin inhibitor (SSI). 1The abbreviations used are: SSIStreptomyces subtilisin inhibitorAPIalkaline protease inhibitorsAAPFN-succinyl-l-Ala-Ala-Pro-Phe5-IAF5-iodoacetamidofluoresceinEIreversible enzyme-inhibitor complexEI*isomer of the second enzyme-inhibitor complexEI**irreversible enzyme-inhibitor complexHPLChigh pressure liquid chromatography.1The abbreviations used are: SSIStreptomyces subtilisin inhibitorAPIalkaline protease inhibitorsAAPFN-succinyl-l-Ala-Ala-Pro-Phe5-IAF5-iodoacetamidofluoresceinEIreversible enzyme-inhibitor complexEI*isomer of the second enzyme-inhibitor complexEI**irreversible enzyme-inhibitor complexHPLChigh pressure liquid chromatography. The future development of these inhibitors for their potential application in therapeutics and biocontrol agents will undoubtedly depend on application of kinetic techniques that yield quantitative information about the behavior of the inhibitors. When the structure of inhibitor can be correlated with the true dissociation constants for their enzyme-inhibitor complexes, a systematic approach can be made toward the design of more effective inhibitors for a target enzyme using protein engineering. Delineating the inhibition mechanism and role of the reactive site residues of the inhibitors and understanding the binding efficiency will provide further insight for their potential application. Considering the physiological importance of the serine alkaline proteases and their role in various physiological and biotechnological processes, there is a lacuna in the studies on the kinetics of the mechanism of inhibition by their naturally occurring protein inhibitors. Streptomyces subtilisin inhibitor alkaline protease inhibitor N-succinyl-l-Ala-Ala-Pro-Phe 5-iodoacetamidofluorescein reversible enzyme-inhibitor complex isomer of the second enzyme-inhibitor complex irreversible enzyme-inhibitor complex high pressure liquid chromatography. Streptomyces subtilisin inhibitor alkaline protease inhibitor N-succinyl-l-Ala-Ala-Pro-Phe 5-iodoacetamidofluorescein reversible enzyme-inhibitor complex isomer of the second enzyme-inhibitor complex irreversible enzyme-inhibitor complex high pressure liquid chromatography. Previously we have reported the isolation and purification of an alkaline protease inhibitor (API) from the extracellular culture filtrate of the Streptomyces sp. NCIM 5127 (14.Vernekar J. Ghatge M. Deshpande V. Biochem. Biophys. Res. Commun. 1999; 262: 702-707Crossref PubMed Scopus (45) Google Scholar). The protein has been purified to homogeneity by ammonium sulfate precipitation, preparative polyacrylamide gel electrophoresis, and anion exchange chromatography. The biochemical characterization has revealed that API is a dimeric protein (Mr 28,000) with five disulfide linkages. Chemical modification studies of API and its binding interaction with the alkaline protease from Conidiobolus sp. have revealed the presence of a tryptophan residue in the reactive site and a disulfide bond at or near the reactive site of the inhibitor (15.Vernekar J. Tanksale A. Ghatge M. Deshpande V. Biochem. Biophys. Res. Commun. 2001; 285: 1018-1024Crossref PubMed Scopus (12) Google Scholar). The biochemical and secondary structural analysis of the inhibitor have revealed that the API belongs to the SSI family of inhibitors. Inhibitors belonging to the SSI family follow the standard mechanism of inhibition wherein the inhibitor acts as a highly specific substrate for limited proteolysis by the target enzyme (16.Laskowaski Jr., M. Kato I. Annu. Rev. Biochem. 1980; 49: 593-626Crossref PubMed Scopus (1936) Google Scholar). These inhibitors bind very tightly to enzyme in the manner of a good substrate and are cleaved very slowly. We present the first report of a proteinaceous API exhibiting slow tight binding inhibition against Proteinase K. The steady-state kinetics revealed a two-step inhibition mechanism and the conformational modes observed during the binding of inhibitor to the enzyme were conveniently monitored by fluorescence analysis. The mechanism of inactivation of Proteinase K by API was delineated and the kinetic parameters associated with the enzyme-inhibitor interaction were determined. The role of hydrogen bonding interactions in the inhibition mechanism of Proteinase K was deciphered by investigating the fluorescence of the 5-iodoacetamidofluorescein (IAF)-labeled enzyme and a model for the mechanism of interactions has been proposed. Materials—Purified Proteinase K from T. album limber, 5-iodoacetamidofluorescein, and sAAPF-p-nitroanilide were obtained from Sigma. All other chemicals used were of analytical grade. Microorganism, Growth Conditions, and Purification of API—The Streptomyces sp. was grown in liquid casein-starch medium for 96 h at 28 °C on a rotary shaker at 200 rpm. The cells were separated by centrifugation and the cell-free supernatant was checked for the presence of API. API was purified from the Streptomyces sp. as reported (14.Vernekar J. Ghatge M. Deshpande V. Biochem. Biophys. Res. Commun. 1999; 262: 702-707Crossref PubMed Scopus (45) Google Scholar). Briefly, the extracellular culture filtrate containing API was concentrated by ultrafiltration and purified by polyacrylamide gel electrophoresis using the gel x-ray film contact print technique. In the gel x-ray film contact print technique, after electrophoretic resolution of the protein, a vertical strip of the gel was cut and incubated for 10 min in 0.1 m carbonate-bicarbonate buffer, pH 10.0, containing 0.5 mg/ml of the bacterial alkaline protease, subtilisin. The gel was overlaid on an equal sized x-ray film and the hydrolysis of gelatin was followed for 20 min at 37 °C. The band corresponding to the inhibitory activity was excised and eluted by homogenization followed by vacuum filtration and purified further by DEAE-cellulose chromatography. Proteinase K Assay and Inhibition Kinetics—Proteolytic activity of Proteinase K was measured by assaying the enzyme activity using casein and the synthetic substrate sAAPF-p-nitroanilide. Proteinase K (100 nm) was dissolved in 0.05 mTris-HCl buffer, pH 8.5, containing 1 mm CaCl2. The reaction was initiated by the addition of 1 ml of casein (5 mg/ml) at 37 °C for 30 min. The reaction was quenched by the addition of 2 ml of 5% acidified trichloroacetic acid followed by incubating for 30 min at room temperature. The unhydrolyzed casein precipitate was separated by centrifugation and filtration through Whatman No. 1 filter paper. Absorbance of the trichloroacetic acid-soluble products was measured at 280 nm. The enzymatic activity in the presence of the synthetic substrate is determined as described (17.DelMar E.G. Largmann C. Brodrick J.W. Geokas M.C. Anal. Biochem. 1979; 99: 316-320Crossref PubMed Scopus (541) Google Scholar). One unit of protease activity was defined as the amount of enzyme that causes an increase of 1 absorbance unit at 280 nm for alkaline proteases. One protease inhibitor unit was defined as the amount of inhibitor that inhibits 1 unit of protease activity. For initial kinetic analysis, the kinetic parameters for the substrate hydrolysis were determined by measuring the initial rate of enzymatic activity. The inhibition constant (Ki) was determined by Dixon (18.Dixon M. Biochem. J. 1953; 55: 170-171Crossref PubMed Scopus (3280) Google Scholar) and using the Lineweaver-Burk method. The Km value was also calculated from the double-reciprocal equation by fitting the data into Microcal Origin. For the Lineweaver-Burk analysis, Proteinase K (1 μm) was incubated with API at 1 and 2.5 μm and assayed at increasing concentrations of casein (1–10 mg/ml). In the method of Dixon (18.Dixon M. Biochem. J. 1953; 55: 170-171Crossref PubMed Scopus (3280) Google Scholar), proteolytic activity of Proteinase K (1 μm) was measured in the presence of 5 and 10 mg/ml of casein, at concentrations of API ranging from 1 to 5 μm at 37 °C for 30 min. The reciprocals of substrate hydrolysis (1/v) were plotted against the inhibitor concentration and the Ki was determined by fitting the data using Microcal Origin. For progress curve analysis, 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). Reaction was initiated by the addition of Proteinase K at 37 °C and the release of products was monitored at different time intervals at 280 nm. In each slow binding inhibition experiment, five to six assays were performed with appropriate blanks. For the kinetic analysis and rate constant determinations, the assays were carried out in triplicate and the average value was considered throughout. Further details of the experiments are given in the respective figure legends. Evaluation of Kinetic Parameters—Initial rate studies for reversible, competitive inhibition were analyzed as per Equation 1.v=VmaxSKm(1+I/Ki)+S(Eq. 1) Where Km is the Michaelis constant, Vmax is the maximal catalytic rate at saturating substrate concentration [S], Ki = (k4/k3) is the dissociation constant for the first reversible enzyme-inhibitor complex, and I is the inhibitor concentration (19.Cleland W.W. Methods Enzymol. 1979; 63: 103-138Crossref PubMed Scopus (1929) Google Scholar). The progress curves for the interactions between API and Proteinase K were analyzed using Equation 2 (20.Beith J.G. Methods Enzymol. 1995; 248: 59-84Crossref PubMed Scopus (186) Google Scholar, 21.Morison J.F. Stone S.R. Comments Mol. Cell. Biophys. 1985; 2: 347-368Google Scholar).[P]=vst+v0-vsk(1-e-kt)(Eq. 2) Where [P] is the product concentration at any time t, v0 and vs are the initial and final steady-state rates, respectively, and k is the apparent first-order rate constant for the establishment of the final steady-state equilibrium. Corrections have been made for the reduction in the inhibitor concentration that occurs on formation of the enzyme inhibitor (EI) complex. This is because in the case of tight binding inhibition, the concentration of EI is not negligible in comparison to the inhibitor concentration and the free inhibitor concentration is not equal to the added concentration of the inhibitor. The corrections of the variation of the steady-state velocity with the inhibitor concentrations were made according to Equations 3 and 4 as described by Morrison and Walsh (22.Morisson J.F. Walsh C.T. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 201-301PubMed Google Scholar).vs=k7SQ2(Km+S)(Eq. 3) Q=[(Ki'+It-Et)+4Ki'Et]1/2-(Ki'+It-Et)(Eq. 4) Where Ki′ = Ki* (1 + S/Km), k7 rate constant for the product formation, and It and Et stand for total inhibitor and enzyme concentrations, respectively. The relationship between the rate constant of enzymatic reaction k, and the kinetic constants for the association and dissociation of the enzyme and inhibitor was determined as per Equation 5.k=k6+k5[I/Ki1+(S/Km)+(I/Ki)](Eq. 5) The progress curves were analyzed by Equations 2 and 5 using non-linear least-square parameter minimization to determine the best-fit values with the corrections for the tight binding inhibition. The overall inhibition constant was determined as given by Equation 6.Ki*=[E][I][EI]+[EI*]=Ki[k6k5+k6](Eq. 6) For the time-dependent inhibition, there exists a time range in the progress curves wherein formation of EI* is small. Within this time range it is possible to measure the effect of the inhibitor on v0, i.e. to measure Ki directly. Values for Ki were obtained from Dixon analysis at a constant substrate concentration as described in Equation 7.1v=1Vmax+KmVmaxS(1+I/Ki)(Eq. 7) The rate constant k6, for the dissociation of the second enzyme-inhibitor complex was measured directly from the time-dependent inhibition. Concentrated Proteinase K and API were incubated in a reaction mixture to reach equilibrium, followed by large dilutions in assay mixtures containing near saturating substrate. Proteinase K (1 mm) was preincubated with API (500 μm) for 30 min in 0.05 m sodium phosphate buffer, pH 7.5. 5 μl of the preincubated sample was removed and diluted 5000-fold in the same buffer and assayed at 50 °C using casein (150 mg/ml) at different time intervals. Gel Filtration Analysis of the Enzyme-Inhibitor Complexes—The quaternary structure of the enzyme-inhibitor complexes was monitored by size exclusion chromatography on a Protein-Pak 300SW HPLC column (7.8 × 300 mm) using a Waters liquid chromatograph system. The column was equilibrated with 0.05 m potassium phosphate buffer, pH 7.5, before the analysis. For the formation of enzyme-inhibitor complex Proteinase K (1 mm) was preincubated with API (500 μm) in 0.05 m sodium phosphate buffer, pH 7.5. 5 μl of the preincubated sample was removed at 30 and after 60 min and loaded on the system to analyze the conformational changes in the enzyme-inhibitor complex. The elution of the complexes was monitored by absorption at 280 nm. Fluorescence Analysis—Fluorescence measurements were performed on a PerkinElmer LS50 luminescence spectrometer. Tryptophanyl fluorescence was excited at 295 nm and emission was recorded from 300 to 500 nm at 25 °C. The slit widths on both the excitation and emission were set at 5 nm and the spectra were obtained at 50 nm/min. For inhibitor binding studies, Proteinase K (1 μm) was dissolved in 0.05 m sodium phosphate buffer, pH 7.5. Titration of the enzyme with API was performed by adding different concentrations of the inhibitor to a fixed concentration of enzyme solution. For each inhibitor concentration on the titration curve a new enzyme solution was used. All the data on the titration curve were corrected for dilutions and the graphs were smoothened. The magnitude of the rapid fluorescence decrease (F0 – F) occurring at each API concentration was computer fitted to Equation 8, to determine the calculated value of Ki and ΔFmax (23.Houtzager V. Oullet M. Falgueyret J.-P. Passmore L.A. Bayly C. Percival M.D. Biochemistry. 1996; 35: 10974-10984Crossref PubMed Scopus (35) Google Scholar).(F0-F)=ΔFmax/{1+(Ki/[I])}(Eq. 8) The first-order rate constants kobs at each inhibitor concentration [I] were fitted to Equation 9 for the determination of k5 under the assumption that for a tight binding inhibitor k6 can be considered negligible at the onset of the slow loss of fluorescence.kobs=k5[I]/{Ki+[I]}(Eq. 9) Time course of protein fluorescence following the addition of inhibitor were measured for 10 min with excitation and emission wavelengths fixed at 295 and 335 nm, respectively, with data acquisition at 0.1-s intervals. Corrections for the inner filter effect were performed as described by Equation 10 (24.Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Press, New York1983: 180-195Google Scholar).Fc=Fantilog[(Aex+Aem)/2](Eq. 10) Where Fc and F stand for the corrected and measured fluorescence intensities, respectively, and Aex and Aem are the absorbence of the solution at the excitation and emission wavelengths, respectively. Background fluorescence from API and buffer were appropriately correct. Fluorescent Chemoaffinity Labeling of Proteinase K and the Effect of API Binding on the 5-IAF Fluorescence—Proteinase K (1 mm) was modified by treatment with 100-fold molar excess of 5-IAF in 50 mm potassium phosphate buffer, pH 7.5, for 24 h in the dark. At pH 7.5, 5-IAF specifically reacts with free –SH groups. The labeled protein was purified through Sephadex G-10 and eluted with phosphate buffer. Fractions showing absorbance at 280 and 490 nm were pooled and concentrated by lyophilization. The fluorescence spectrum of 5-IAF-labeled Proteinase K was monitored at an excitation wavelength of 490 nm and emission wavelength from 490 to 600 nm. The number of Cys residues labeled was determined by using the molar absorptivity for 5-IAF of 80,000 cm–1m–1. The stoichiometry of the fluorescence labeling was determined to be 1:1 between 5-IAF and Proteinase K. Kinetic Analysis of the Inhibition of Proteinase K by API— The API was produced extracellularly by a Streptomyces sp., and has been characterized for its inhibitory activity toward the alkaline proteases (14.Vernekar J. Ghatge M. Deshpande V. Biochem. Biophys. Res. Commun. 1999; 262: 702-707Crossref PubMed Scopus (45) Google Scholar). Initial inhibition kinetic assessments revealed that API is a competitive inhibitor of Proteinase K with an IC50 (concentration of the inhibitor required for 50% inhibition of the enzyme) value of 5.5 ± 0.5 × 10–5m (Fig. 1). The steady-state rate of proteolytic activity of Proteinase K was reached rapidly in the absence of API, whereas, in its presence a time-dependent decrease in the rate as a function of the inhibitor concentration was observed. The progress curves in the presence of API revealed a time range where the initial rate of reaction was similar to that in the absence of the inhibitor, and does not deviate from linearity (Fig. 2), therefore conversion of EI to EI* was minimal. This time range for a low concentration of API was 8 min for Proteinase K, wherein all the classical competitive inhibition experiments were used to determine the Ki (k4/k3) value (Equation 5). The inhibition constant Ki associated with the formation of the reversible EI determined from the reciprocal equation was 5.2 ± 0.6 × 10–6m, which was further corroborated by the Dixon method (Fig. 3). The apparent rate constant k, derived from the progress curves of Proteinase K when plotted versus the inhibitor concentration followed a biphasic hyperbolic function (Fig. 4), revealing that fast equilibrium precedes the formation of the final slow dissociating enzyme-inhibitor complex (EI*), indicating a two-step, slow tight inhibition mechanism. Indeed the data could be analyzed with Equation 5 by non-linear regression analysis to obtain the best estimate of the overall inhibition constant Ki* (2.5 ± 0.3 × 10–7m). In the case of slow tight binding inhibitors, because the EI* complex is stable and dissociates slowly, the rate constant k6, for the conversion of EI* to EI was determined in an alternative method, by pre-incubating high concentrations of enzyme and inhibitor for sufficient time to allow the system to reach equilibrium. Furthermore, large dilution of the enzyme-inhibitor complex into a relatively large volume of assay mixture containing saturating substrate concentrations causes dissociation of the enzyme-inhibitor complex and the dissociation constant can be determined by the regeneration of enzymatic activity. Under these conditions, v0 and the effective inhibitor concentration are considered approximately equal to zero and the rate of activity regeneration will provide the k6 value. The preincubated (30 min) mixture of Proteinase K and API was diluted 5,000-fold into the assay mixture containing the substrate at a saturating substrate concentration. By least-squares minimization of Equation 2 to the data for recovery of enzymatic activity, the determined k6 value was 4.5 ± 0.5 × 10–5 s–1 (Fig. 5), which clearly indicated a very slow dissociation of EI*. The final steady-state rate vs was determined from the control that was preincubated without the inhibitor. The value of the rate constant k5, associated with the isomerization of EI to EI*, was 9.2 ± 1 × 10–3 s–1 as obtained from fits of Equation 3 to the onset of inhibition data using the experimentally determined values of Ki and k6 (Table I). The overall inhibition constant Ki* is a function of k6/(k5 + k6) and is equal to the product of Ki and this function. The k6 value indicated a slower rate of dissociation of EI* and the half-life (t½) for the reactivation of EI* determined from the k6 value was 4.27 ± 0.5 h, suggesting higher binding affinity of API toward Proteinase K. When the incubation time was more than 60 min, the inhibitor failed to dissociate from the complex, because there was no recovery in the enzymatic activity (Fig. 5). This observation can be attributed to gross conformational changes in the EI* complex induced by leading toward the formation of a conformationally locked irreversible enzyme-inhibitor complex (EI**). To characterize the EI** we have carried out the quaternary structural analysis by gel filtration chromatography on a HPLC system. As revealed by Fig. 6A, the complex formed after 30 min of equilibration showed a retention time of 16 min, which demonstrated the formation of the EI* complex. However, when the equilibration time between the enzyme and inhibitor was more than 60 min (Fig. 6B), the retention time of the complex was shifted to 15 min indicating a change in the conformation of the EI*. This difference in the retention time can be attributed to the formation of irreversible complex EI** because of gross conformational changes induced in the EI* complex. These conformational states of the enzyme-inhibitor complex are further characterized by fluorescence spectroscopy. All our kinetic analysis for the slow tight binding inhibition has been determined in the time frame before the formation of the irreversible complex.Fi
Referência(s)