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1.
The theory of acyl-enzyme kinetics (Bender, M.L., Kézdy, F.J. and Wedler, F.C. (1967) J. Chem. Educ. 44, 84-88) has been adapted for use in evaluating the kinetics of inhibition of serine proteases by both natural and synthetic irreversible inhibitors. The new theory is based upon formal analysis of the case of an irreversible, active-site-directed inhibitor competing with an irreversible, active-site-directed substrate for the active site of a serine protease. From this theory, an experimentally simple and accurate method is described to obtain a second-order rate constant that is characteristic of the efficiency with which an irreversible inhibitor reacts. The experimental method is particularly useful for characterizing fast-acting, irreversible inhibitors. The theory and method which are applicable to a wide variety of enzymes are verified by analysis of the inhibition of bovine trypsin by three model inhibitors, p-nitrophenyl p'-guanidinobenzoate, soybean trypsin inhibitor and alpha-1-proteinase inhibitor as well as by human antithrombin III in the presence of heparin and by bovine pancreatic trypsin inhibitor.  相似文献   

2.
The reaction of chymase, a chymotryptic proteinase from human skin, and bovine pancreatic chymotrypsin with a number of time-dependent inhibitors has been studied. An integrated equation, relating product formation with time, has been derived for the reaction of enzymes with time-dependent inhibitors in the presence of substrate. This is based on a two-step model in which a rapidly reversible, non-covalent complex (EI) is formed prior to a tighter, less readily reversible complex (EI)*). The equation depends on the simplifying assumption [I] much greater than [E], but is applicable to reversible and irreversible slow-binding and tight-binding inhibitors whether or not they show saturation kinetics. The method has been applied to the reaction of chymase and chymotrypsin with the tetrapeptide aldehyde, chymostatin, basic pancreatic trypsin inhibitor and Ala-Ala-Phe-chloromethylketone (AAPCK). The irreversible inhibitor, AAPCK, showed the expected saturation kinetics for both enzymes and the apparent first-order rate constants (k2) and dissociation constants (Ki) for the non-covalent complexes were determined. Chymostatin was a much more potent inhibitor which failed to show a saturation effect. The second-order rate constant of inactivation (k2/Ki), the first-order reactivation rate constant (k-2), and the dissociation constant of the covalent complex (Ki*) were determined. Basic pancreatic trypsin inhibitor, a potent inhibitor of chymotrypsin, had similar kinetics to chymostatin but failed to inhibit chymase. The applicability of the two-step model and the integrated equation to slow- and tight-binding inhibitors is discussed in relation to a number of examples from the literature.  相似文献   

3.
In vivo significance of kinetic constants of protein proteinase inhibitors   总被引:7,自引:0,他引:7  
We describe the in vivo significance of the kinetic parameters which characterize the interaction between proteinases and protein proteinase inhibitors. Knowledge of the second-order association rate constant kass and in vivo inhibitor concentration allows the calculation of the delay time of inhibition, i.e., the time required for complete inhibition of a proteinase in vivo. The influence of biological substrates on the delay time is also analyzed. The extent of substrate breakdown during the delay time of inhibition may be computed from the various constants describing the proteinase/substrate/inhibitor interactions and the biological concentrations of proteinase and inhibitor. The in vivo partition of a proteinase between two inhibitors may be calculated if the kinetic parameters are known. We define a stability time for enzyme-inhibitor complexes as a minimal time during which the complexes may be considered as stable. This time is related to kdiss the dissociation rate constant of the reversible enzyme-inhibitor complex or to k, the breakdown rate constant of the complex formed with temporary inhibitors. The overall stability of the complex depends upon the ratio between the inhibitor concentration and Ki, the equilibrium dissociation constant of the complex. If this ratio is higher than 1000, a reversible inhibitor behaves like an irreversible one in vivo whatever the enzyme concentration.  相似文献   

4.
Most proteinase inhibitors from plant seeds are assumed to contribute to broad-spectrum protection against pests and pathogens. In oat (Avena sativa L.) grain the main serine proteinase inhibitors were found to be serpins, which utilize a unique mechanism of irreversible inhibition. Four distinct inhibitors of the serpin superfamily were detected by native PAGE as major seed albumins and purified by thiophilic adsorption and anion exchange chromatography. The four serpins OSZa-d are the first proteinase inhibitors characterized from this cereal. An amino acid sequence close to the blocked N-terminus, a reactive centre loop sequence, and the second order association rate constant (ka') for irreversible complex formation with pancreas serine proteinases at 24 degrees C were determined for each inhibitor. OSZa and OSZb, both with the reactive centre scissile bond P1-P1' Thr downward arrow Ser, were efficient inhibitors of pancreas elastase (ka' > 105M-1 s-1). Only OSZb was also an inhibitor of chymotrypsin at the same site (ka' = 0.9 x 105M-1 s-1). OSZc was a fast inhibitor of trypsin at P1-P1' Arg downward arrow Ser (ka' = 4 x 106M-1 s-1); however, the OSZc-trypsin complex was short-lived with a first order dissociation rate constant kd = 1.4 x 10-4 s-1. OSZc was also an inhibitor of chymotrypsin (ka' > 106M-1 s-1), presumably at the overlapping site P2-P1 Ala downward arrow Arg, but > 90% of the serpin was cleaved as substrate. OSZd was cleaved by chymotrypsin at the putative reactive centre bond P1-P1' Tyr downward arrow Ser, and no inhibition was detected. Together the oat grain serpins have a broader inhibitory specificity against digestive serine proteinases than represented by the major serpins of wheat, rye or barley grain. Presumably the serpins compensate for the low content of reversible inhibitors of serine proteinases in oats in protection of the grain against pests or pathogens.  相似文献   

5.
This work presents a kinetic study on irreversible inhibition of trypsin by TLCK, using a new experimental approach. The method consists of the incubation of the enzyme with an irreversible inhibitor in the presence of a substrate which allows enzyme turnover as well as continuous measurement of the appearance of the product, a simultaneous change in the initial concentrations of the irreversible inhibitor and enzyme being undertaken, though a constant ratio between the latter, is maintained. This new approach enables the kinetic constants for TLCK, k2 and K1, to be determined.  相似文献   

6.
The turkey reproductive tract and seminal plasma contain a serine proteinase inhibitor that seems to be unique for the reproductive tract. Our experimental objective was to isolate, characterize and cDNA sequence the Kazal family proteinase inhibitor from turkey seminal plasma and testis. Seminal plasma contains two forms of a Kazal family inhibitor: virgin (Ia) represented by an inhibitor of moderate electrophoretic migration rate (present also in the testis) and modified (Ib, a split peptide bond) represented by an inhibitor with a fast migration rate. The inhibitor from the seminal plasma was purified by affinity, ion-exchange and reverse phase chromatography. The testis inhibitor was purified by affinity and ion-exchange chromatography. N-terminal Edman sequencing of the two seminal plasma inhibitors and testis inhibitor were identical. This sequence was used to construct primers and obtain a cDNA sequence from the testis. Analysis of a cDNA sequence indicated that turkey proteinase inhibitor belongs to Kazal family inhibitors (pancreatic secretory trypsin inhibitors, mammalian acrosin inhibitors) and caltrin. The turkey seminal plasma Kazal inhibitor belongs to low molecular mass inhibitors and is characterized by a high value of the equilibrium association constant for inhibitor/trypsin complexes.  相似文献   

7.
The kinetics of the irreversible inhibition of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) by diisopropyl fluorophosphate and paraoxon have been studied by the approach of following the substrate reaction continuously in the presence of both the substrate and the inhibitor based on kinetic equations previously derived (Tsou, C.-L. (1965) Acta Biochim. Biophys. Sinica 5, 387-417). From determinations of the effects of different concentrations of substrate and the inhibitors on the apparent rate constants for the irreversible inhibition reactions it can be shown that these inhibitors are of the competitive complexing type. Both the reversible dissociation constant for the enzyme inhibitor complex and the rate constant for the subsequent phosphorylation step can be obtained from suitable plots of the experimental data.  相似文献   

8.
A new simple graphical method is described for the determination of inhibition type and kinetic parameters of an enzyme reaction without any replot. The method consists of plotting experimental data as v/(vo--v) versus the reciprocal of the inhibitor concentration at different substrate concentrations, where v and vo represent the velocity in the presence and in the absence of the inhibitor respectively with a given concentration of the substrate. Partial inhibition gives straight lines that converge on the abscissa at a point away from the origin, whereas complete inhibition gives lines that go through the origin. The inhibition constants of enzymes and the reaction rate constant of the enzyme-substrate-inhibitor complex can be calculated from the abscissa and ordinate intercepts of the plot. The relationship between the slope of the plot and the substrate concentration shows characteristic features depending on the inhibition type: for partial competitive inhibition, the straight line converging on the abscissa at--Ks, the dissociation constant of the enzyme-substrate complex; for non-competitive inhibition, a constant slope independent of the substrate concentration; for uncompetitive inhibition, a hyperbola decreasing with the increase in the substrate concentration; for mixed-type inhibition, a hyperbola increasing with the increase in the substrate concentration. The properties of the replot are useful in confirmation of the inhibition mechanism.  相似文献   

9.
The efflux of a substrate from preloaded cells may be decelerated by an inhibitor in the external medium or accelerated by a compatible substrate in the external medium. The derivations of rate equations for the initial velocity of the zero-trans reaction, trans efflux inhibition, and accelerated exchange diffusion are described for steady state conditions. The rate constants making up the Michaelis constant for the trans inhibition reaction are the same as the corresponding parameters in the zero-trans reaction. The rate constants making up the Michaelis constant for the accelerated exchange reaction, however, are different from the corresponding parameters in the zero-trans reaction. The rate equation for trans inhibition shows that the velocity constant for recovery of the unloaded carrier may be determined with steady state experimental data. It is suggested that the observed recovery constant is independent of the substrates and trans inhibitors chosen for an assay of a particular carrier system. An experiment is briefly described to show a determination of a tentative value for the recovery constant of the unloaded nucleoside carrier in yeast cells and the apparent inhibition constant for a trans inhibitor.  相似文献   

10.
Mechanism of action of inter-alpha-trypsin inhibitor   总被引:1,自引:0,他引:1  
C W Pratt  S V Pizzo 《Biochemistry》1987,26(10):2855-2863
Inter-alpha-trypsin inhibitor (I alpha I) is a unique proteinase inhibitor that can be proteolyzed by the same enzymes that are inhibited, to generate smaller inhibitors. This study examines the reactions of I alpha I with trypsin, chymotrypsin, plasmin, and leukocyte elastase. Complexes of I alpha I and proteinase were demonstrated by gel filtration chromatography. Complete digestion of I alpha I by each proteinase was not accompanied by a comparable loss of inhibition of that enzyme or a different enzyme. Following proteolysis, inhibitory activity was identified in I alpha I fragments of molecular weight 50,000-100,000 and less than 40,000. Addition of a second proteinase inhibitor prevented proteolysis. Both I alpha I and its complex with proteinase were susceptible to degradation. Kinetic parameters for both the inhibition and proteolysis reactions of I alpha I with four proteinases were measured under physiological conditions. On the basis of these results, a model for the mechanism of action of I alpha I is proposed: Proteinase can react with either of two independent sites on I alpha I to form an inhibitory complex or a complex that leads to proteolysis. Both reactions occur simultaneously, but the inhibitory capacity of I alpha I is not significantly affected by proteolysis since the product of proteolysis is also an inhibitor. For a given proteinase, the inhibition equilibrium constant and the Michaelis constant for proteolysis describe the relative stability of the inhibition and proteolysis complexes; the second-order rate constants for inhibition and proteolysis indicate the likelihood of either reaction. The incidence of inhibition or proteolysis reactions involving I alpha I in vivo cannot be assessed without knowledge of the exact concentrations of inhibitor and proteinases; however, analysis of inhibition rate constants suggests that I alpha I might be involved in plasmin inhibition.  相似文献   

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