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PubMed1981

31P NMR study of the kinetics of binding of myo-inositol hexakisphosphate to human hemoglobin. Observation of fast-exchange kinetics in high-affinity systems.

The association and dissociation kinetics of the complexes of myo-inositol hexakisphosphate (P6-inositol) with deoxyhemoglobin (Hb) and carboxyhemoglobin (HbCO) have been investigated by 31P NMR between pH 6.8 and pH 5.5. These complexes represent high-affinity systems with binding constants varying between 10(5) M-1 and 2 X 10(9) M-1. 31P NMR spectra of P6-inositol were recorded in the presence of hemoglobin as a function of the P6-inositol/hemoglobin molar ratio. It appeared that the exchange of the polyphosphate molecule between the solution and the central cavity binding site is fast on the NMR time scale. This observation cannot be reconciled with a single-step binding mechanism of P6-inositol to hemoglobin. Analysis of the spectra revealed the occurrence of additional binding of P6-inositol to both Hb and HbCO. This binding was also observed in pH-state experiments performed at low ionic strength. 31P NMR experiments carried out with hemoglobin of which the alpha-chain N termini were carbamylated, strongly suggest that these termini constitute the additional binding site for P6-inositol. A model is proposed which accounts for the enhancement of exchange kinetics in these high-affinity systems. In this model a rapid migration is assumed for P6-inositol between the central cavity binding site and an entry/leaving site on the hemoglobin molecule. Based on this model 31P NMR linewidths and chemical shift patterns for this three-site exchange problem were calculated.

Carboxyhemoglobin↗
PubMed1996

Slow-binding inhibition of gamma-aminobutyric acid aminotransferase by hydrazine analogues.

(3-Hydroxybenzyl)hydrazine and methylhydrazine have been found to be potent slow-binding inhibitors of the pyridoxal 5-phosphate (PLP)-dependent enzyme gamma-aminobutyric acid aminotransferase (GABA-AT). Both compounds follow mechanism A (Morrison, J.F.; Walsh, C. T. Adv. Enzymol. 1988, 61, 201-301) which does not involve formation of a rapidly reversible enzyme-inhibitor complex before the formation of the final tight complex. The rate constant for formation of the enzyme-inhibitor complex determined from the slow-binding kinetics was 2.08 x 10(3) and 1.98 x 10(4) M-1 min-1 for methylhydrazine and (3-hydroxybenzyl)hydrazine, respectively. The rate constant for dissociation of the enzyme--inhibitor complex determined for the slow-binding kinetics was 4.6 x 10(-3) and 5 x 10(-3) min-1, respectively. The inhibition constants calculated from the slow-binding inhibition kinetics are 2.2 microM for methylhydrazine and 0.3 microM for (3-hydroxybenzyl)hydrazine. Reactivation of the inhibited enzyme was not first order, perhaps due to a side reaction of the hydrazine, but was consistent with the results obtained from the slow-binding kinetics. Inhibition constants were calculated from the level of enzyme activity at equilibrium inhibition. These constants are 2.8 and 0.46 microM for methylhydrazine and (3-hydroxybenzyl)hydrazine, respectively, in good agreement with those calculated from the slow-binding inhibition kinetics. 3-Hydrazinopropionate also behaved as a slow-binding inhibitor. However, the dependence of its kinetics on the concentration of inhibitor could not be described by the slow-binding or slow, tight-binding inhibition models. These kinetics could not be described by the tight-binding character of the inhibition because the addition of the competitive inhibitor propionic acid at 100 times its Ki did not affect the shape of the curve for inhibitor concentration dependence. The slow-binding inhibition appeared to require 2-4 molecules of 3-hydrazinopropionate/enzyme. The reactivation of enzyme inhibited by 3-hydrazinopropionate was first order with a rate constant of 6.9 x 10(-3) min-1. Its equilibrium inhibition constant was calculated to be < 20 nM. However, the inhibition constant calculated was dependent on the concentration of inhibitor because of the unusual character discussed above and may be much lower. Only 1 PLP/enzyme dimer reacted with methylhydrazine or (3-hydroxybenzyl)hydrazine, as indicated by Scatchard plots, or with 3-hydrazinopropionate, as shown by a spectrophotometric titration. Slow-binding inhibition does not appear to be the result of a significant enzyme conformational change because there is no change in the tryptophan fluorescence of GABA-AT upon binding either methylhydrazine or 3-hydrazinopropionate. Implications for the design of hydrazine inhibitors of GABA-AT are discussed.

4-Aminobutyrate Transaminase↗
PubMed1998

The domain structure of ICAM-1 and the kinetics of binding to rhinovirus.

Fragments of intercellular adhesion molecule 1 (ICAM- 1) containing only the two most N terminal of its five immunoglobulin SF domains bind to rhinovirus 3 with the same affinity and kinetics as a fragment with the entire extracellular domain. The fully active two-domain fragments contain 5 or 14 more residues than a previously described fragment that is only partially active. Comparison of X-ray crystal structures show differences at the bottom of domain 2. Four different glycoforms of ICAM- 1 bind with identical kinetics.

Antibodies, Monoclonal↗
PubMed1976

The kinetics of binding of U-U-C-A to a dodecanucleotide anticodon fragment from yeast tRNA-Phe.

The kinetics of U-U-C-A binding to the dodecanucleotide (A-Cm-U-Gm-A-A-Y-A-psi-m5C-U-Gp) isolated from the anticodon region of yeast tRNA-Phe are similar to the kinetics of binding of U-U-C-A to intact tRNA-Phe. A large enhancement in binding constant over that predicted for U-U-C-A-U-G-A-A is observed for both the complexes of dodecanucleotide and tRNA-Phe with U-U-C-A. This strongly suggests that both the anticodon loop in tRNA-Phe and the dodecanucleotide can form four base pairs with U-U-C-A. Furthermore, the enhanced stability cannot be attributed to a special conformation of the anticodon loop, but instead the anticodon loop is probably flexible. A likely explanation for the increased binding is the effect of non-base-paired ends. This increased thermodynamic stability comes from a larger entropy gain rather than a larger enthalpy decrease.

Adenine Nucleotides↗
PubMed1996

Human constant regions influence the antibody binding characteristics of mouse-human chimeric IgG subclasses.

Although antibody affinity is primarily determined by immunoglobulin variable region structure human IgG antibodies of the four subclasses specific for the same antigen have been shown to differ in their affinity. To explore the influence of the immunoglobulin constant region on functional antibody affinity, a set of V region identical mouse-human chimeric IgG subclasses specific for TAG72 (tumour-associated glycoprotein) were studied. Biomolecular interaction analysis (BIA) was used to determine the binding kinetics of whole IgG subclasses and F(ab')2 fragments. Despite identical V regions, binding kinetics differed for the four subclasses. The apparent dissociation rate constants of the intact immunoglobulins ranked IgG4 < IgG3 < IgG2 < IgG1. In contrast, analysis of the binding characteriztics of the F(ab')2 fragments derived from IgG1, IgG2 and IgG4 revealed identical binding kinetics. The structure of the constant regions of the humanized IgG subclass antibodies clearly influenced functional antibody affinity, as has been described for the murine IgG subclasses. The exact mechanism for this phenomenon remains obscure but such differences should be taken into account when designing or choosing antibodies for therapeutic use.

Animals↗
PubMed1975

Kinetics of binding of carbon monoxide to lumbricus erythrocruorin: a possible model.

This paper represents a kinetic study of the binding of carbon monoxide by Lumbricus erythrocruorin. Observations on the quantum and the relaxation of the system both to equilibrium and to the steady state realized in the presence of constant illumination under various conditions are reported. The results, besides indicating the existence of at least two types of binding sites, give indications as to the behavior of a complex polyfunctional molecule, such as an enzyme, working under steady-state conditions.

Animals↗
PubMed1993

Kinetics of binding and hydrolysis of a series of nucleoside triphosphates by actomyosin-S1. Relationship between solution rate constants and properties of muscle fibers.

We have measured the steady state kinetics of hydrolysis and presteady state kinetics of binding of the nucleoside triphosphate GTP, CTP, aza-ATP (1-N6-etheno-2-aza-ATP), and ATP by rabbit skeletal actomyosin-S1. The maximum rates of steady state hydrolysis at 10 degrees C at low ionic strength are: CTP, 1.9 s-1 > ATP, 1.3 s-1 > aza-ATP, 0.19 s-1 > GTP, 0.03 s-1. A similar dependence of the rate of steady state hydrolysis upon nucleotide structure has been observed in isometrically contracting muscle fibers in the accompanying paper (Pate, E., Franks-Skiba, K., White, H., and Cooke, R. (1993) J. Biol. Chem. 268, 10046-10053) which strongly suggests that the same biochemical step that limits the maximum rate of hydrolysis of nucleoside triphosphates by actomyosin-S1 in solution also limits the rate of hydrolysis by isometrically contracting muscle fibers. The apparent second order rate constants for the dissociation of actomyosin-S1 by nucleoside triphosphates at 10 degrees C are: ATP, 2.7 x 10(6) M-1 s-1 > aza-ATP, 3.4 x 10(5) M-1 s-1 > GTP, 2.5 x 10(5) M-1 s-1 > CTP, 1.4 x 10(5) M-1 s-1. There is an excellent correlation between the second order rate constant for the dissociation of actomyosin-S1 in solution and the dependence of shortening velocity in glycerinated muscle fibers upon the concentration for ATP, aza-ATP, and CTP (as per accompanying article; Pate et al., 1993). We have used the second order rate constants obtained in solution for the dissociation of actomyosin-S1 by these nucleotides and shortening velocity data obtained with the same nucleoside triphosphates in glycerinated psoas fibers in the accompanying article (Pate et al., 1993) to determine the average distance over which cross-bridges remain attached during unloaded shortening to be 5-12 nm.

Actins↗
PubMed2004

Binding and kinetic mechanisms of the Zeta class glutathione transferase.

The Zeta class of glutathione transferases (GSTs) has only recently been discovered and hence has been poorly characterized. Here we investigate the substrate binding and kinetic mechanisms of the human Zeta class GSTZ1c-1c by means of pre-steady state and steady-state experiments and site-directed mutagenesis. Binding of GSH occurs at a very low rate compared with that observed for the more recently evolved GSTs (Alpha, Mu, and Pi classes). Moreover, the single step binding mechanism observed in this enzyme is reminiscent of that found for the Theta class enzyme, whereas the Alpha, Mu, and Pi classes have adopted a multistep binding mechanism. Replacement of Cys16 with Ala increases the rate of GSH release from the active site causing a 10-fold decrease of affinity toward GSH. Cys16 also plays a crucial role in co-substrate binding; the mutant enzyme is unable to bind the carcinogenic substrate dichloroacetic acid in the absence of GSH. However, both substrate binding and GSH activation are not rate-limiting in catalysis. A peculiarity of the hGSTZ1c-1c is the half-site activation of bound GSH. This suggests a primitive monomer-monomer interaction that, in the recently diverged GSTP1-1, gives rise to a sophisticated cooperative mechanism that preserves the catalytic efficiency of this GST under stress conditions.

Binding Sites↗
PubMed1989

Kinetic study of the slow cyanide binding to Glycera dibranchiata monomer hemoglobin components III and IV.

Compared to other monomeric heme proteins and the heme peroxidases, the Glycera dibranchiata monomer hemoglobin components III and IV exhibit very slow cyanide binding kinetics. This is agreement with the previously reported behavior of component II. Similar to component II, components III and IV have been studied under pseudo-first-order conditions at pH 6.0, 7.0, 8.0, and 9.0 by using a 100-250-fold excess of potassium cyanide at each pH. At 20 degrees C with micromolar protein concentrations, kobs for component III varies between 7.08 x 10(-5) s-1 at pH 6.0 and 100-fold cyanide excess and 1.06 x 10(-2) s-1 at pH 9.0 and 250-fold cyanide excess. For component IV, the values are 2.03 x 10(-4) s-1 for 100-fold cyanide excess at pH 6.0 and 4.13 x 10(-2) s-1 for 250-fold cyanide excess at pH 9.0. In comparison to other heme proteins, our analysis shows that the bimolecular rate constant (klapp) is small. For example, at pH 7.0, it is 3.02 x 10(-1) M-1 s-1 for component III and 1.82 M-1 s-1 for component IV, compared to 400 M-1 s-1 for sperm whale metmyoglobin, 692 M-1 s-1 for soybean metleghemoglobin a, 111 M-1 s-1 for guinea pig methemoglobin, and 1.1 x 10(5) M-1 s-1 for cytochrome c peroxidase. Our results also show that the dissociation rates (k-lapp) are extremely slow and no larger than 10(-6) s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells.

In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, tau TDBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID50D,DBDS,MCD (0.5 +/- 0.1 microM) for the H2-DIDS (4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate) inhibition of tau DBDS is in agreement with the ID50,Cl-MCD (0.94 +/- 0.07 microM) for H2-DIDS inhibition of MCD cell Cl- flux, thus relating tau DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl- exchange by a factor of two, from tau Cl- = 0.30 +/- 0.02 sec to 0.56 +/- 0.06 sec (30 mM NaHCO3). The ID50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003 +/- 0.001 microM, so that binding is about an order of magnitude tighter than that for inhibition of rbc K+ flux (KI,K+,rbc = 0.017 microM). These experiments indicate that the Na+,K+-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells. modulates DBDS binding kinetics with a physiological ID50,DBDS,MCD (0.076 +/- 0.005 microM); 2 microM CE also more than doubles the Cl- exchange time constant from 0.20 +/- 0.04 sec to 0.50 +/- 0.08 sec (30 mM NaHCO3). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1979

Kinetic and binding properties of the oxoglutarate translocator of rat-heart mitochondria.

The kinetic study of the oxoglutarateout/malatein exchange through the inner mitochondrial membrane of rat-heart mitochondria has been compelted and extended to higher external-oxoglutarate and to lower internal-malate concentrations. It has been found that the external oxoglutarate inhibits the exchange at high concentration. This excess-substrate inhibition is preceded by four jumps. The kinetic-saturation curve by the internal malate presents an apparent positive cooperativity that may be interpreted in different ways. The independence of the effects of the two substrates on the initial rate has been observed again and supports the conclusions reached in previous work. A method for the determination of oxoglutarate binding to the external face of the inner membrane is described. The binding curve shows four intermediary plateau regions that reflect significant apparent K-effects, alternatively negative and positive. For external-oxoglutarate concentrations below the region of excess-substrate inhibition, the binding-saturation curve and the kinetic-saturation curve are similar, demonstrating that K-effects are predominant. A particularly wide intermediary plateau that seems to correspond to half saturation of the active sites is common to both saturation curves. A clear lack of proportionality between the two curves at low oxoglutarate concentrations seems to indicate that more than one catalytic-rate constant is implied in the exchange kinetics. Two models of the oxoglutarate carrier are presented. Both lead to a minimum degree of 10:10 for the equation of the binding of oxoglutarate to the catalytic sites. In the first model this corresponds to ten subunits associated into a single oligomer while in the second model this results from a mixture of monomeric, dimeric, trimeric and tetrameric associations.

Animals↗
PubMed2003

Interflavin electron transfer in human cytochrome P450 reductase is enhanced by coenzyme binding. Relaxation kinetic studies with coenzyme analogues.

The role of coenzyme binding in regulating interflavin electron transfer in human cytochrome P450 reductase (CPR) has been studied using temperature-jump spectroscopy. Previous studies [Gutierrez, A., Paine, M., Wolf, C.R., Scrutton, N.S., & Roberts, G.C.K. Biochemistry (2002) 41, 4626-4637] have shown that the observed rate, 1/tau, of interflavin electron transfer (FADsq - FMNsq-->FADox - FMNhq) in CPR reduced at the two-electron level with NADPH is 55 +/- 2 s-1, whereas with dithionite-reduced enzyme the observed rate is 11 +/- 0.5 s-1, suggesting that NADPH (or NADP+) binding has an important role in controlling the rate of internal electron transfer. In relaxation experiments performed with CPR reduced at the two-electron level with NADH, the observed rate of internal electron transfer (1/tau = 18 +/- 0.7 s-1) is intermediate in value between those seen with dithionite-reduced and NADPH-reduced enzyme, indicating that the presence of the 2'-phosphate is important for enhancing internal electron transfer. To investigate this further, temperature jump experiments were performed with dithionite-reduced enzyme in the presence of 2',5'-ADP and 2'-AMP. These two ligands increase the observed rate of interflavin electron transfer in two-electron reduced CPR from 1/tau = 11 s-1 to 35 +/- 0.2 s-1 and 32 +/- 0.6 s-1, respectively. Reduction of CPR at the two-electron level by NADPH, NADH or dithionite generates the same spectral species, consistent with an electron distribution that is equivalent regardless of reductant at the initiation of the temperature jump. Spectroelectrochemical experiments establish that the redox potentials of the flavins of CPR are unchanged on binding 2',5'-ADP, supporting the view that enhanced rates of interdomain electron transfer have their origin in a conformational change produced by binding NADPH or its fragments. Addition of 2',5'-ADP either to the isolated FAD-domain or to full-length CPR (in their oxidized and reduced forms) leads to perturbation of the optical spectra of both the flavins, consistent with a conformational change that alters the environment of these redox cofactors. The binding of 2',5'-ADP eliminates the unusual dependence of the observed flavin reduction rate on NADPH concentration (i.e. enhanced at low coenzyme concentration) observed in stopped-flow studies. The data are discussed in the context of previous kinetic studies and of the crystallographic structure of rat CPR.

Cloning, Molecular↗
PubMed1980

Adenosine receptors in brain membranes: binding of N6-cyclohexyl[3H]adenosine and 1,3-diethyl-8-[3H]phenylxanthine.

N6-Cyclohexyl[3H]adenosine ([3H]CHA) and 1,3-diethyl-8-[3H]phenylxanthine ([3H]DPX) to bind to adenosine receptors in brain membranes. The agonist [3H]CHA has high affinity in both bovine and guinea pig brain (Kd, 0.7 nM and 6 nM, respectively). [3H]CHA binding kinetics are slow (dissociation t1/2;60 min); binding is much higher at 25 degrees C than at 0 degrees C and is inhibited by guanine nucleotides. Potencies of nucleosides and xanthines in competing for [3H]CHA sites indicate that specific binding is entirely to A1 adenosine receptors. In bovine brain, the antagonist [3H]DPX exhibits high-affinity binding (Kd, 5 nM) to the same A1 receptors that bind [3H]CHA. Binding kinetics are rapid (dissociation t1/2, 1 min), and binding is moderately higher at 0 degrees C than at 25 degrees C. In guinea pig brain, [3H]DPX binding has only moderate affintiy (Kd 50 nM), and about 60% of specific binding is to sites that resemble A2 adenosine receptors.

Adenosine↗
PubMed2002

Effects of pH on kinetics of binding of mRNA-cap analogs by translation initiation factor eIF4E.

Stopped-flow spectrofluorimetry and a theoretical method for predicting protonation equilibria in polyelectrolytes were combined in an analysis of the pH dependence of the kinetics of binding of analogues of the 5'-mRNA cap to the cap binding protein eIF4E. The computer simulations and available experimental data indicate that there are two titratable groups in the binding site of the protein and two titratable groups on the ligands directly involved in the binding, in addition to stacking interactions described by other groups. The observed pH dependencies of the rate constants obtained from the stopped-flow experiments are consistent with this finding. In particular, it is concluded that binding of both forms of the cap analogs regarding protonation at the N1 position of the guanine ring is efficient, and the shift to a predominantly protonated form of the ring takes place after formation of the complex.

Animals↗
PubMed2005

Variations in antigen-antibody association kinetics as a function of pH and salt concentration: a QSAR and molecular modeling study.

The relationship between three environmental factors (ionic strength, pH, and temperature) and antigen-antibody binding kinetics was investigated using QSAR (quantitative structure-activity relationship) and molecular modeling approaches. The interaction used for this analysis is that between the camel antibody fragment cAbLys3 and lysozyme. Binding kinetics were measured using a Biacore 2000 instrument, at NaCl concentrations between 50 and 500 mM, at pH's between 5 and 10, and at temperatures between 15 and 30 degrees C, according to multivariate experimental designs. Variations in kinetic on- and off-rate parameters were up to 400- and 16-fold, respectively. Mathematical models that relate log k(on) to experimental conditions were developed. They indicated an influence of all three factors, with a clear dependency between pH and NaCl concentration for their effect on k(on). These models were able to predict on-rate parameters under new experimental conditions. Titration calculations using continuum electrostatics were performed on the crystallographic structures of the isolated and bound proteins to gain structural insight for the on-rate enhancement observed at pH <6.5 and low salt concentrations. These calculations rule out electrostatic steering linked to global and/or local charge variations in the molecules as the factor responsible for the on-rate enhancement at low pH. His 111 of cAbLys3, located at the binding interface, can adopt two side chain orientations with different intramolecular contacts. The results of the calculations suggest an alternative mechanism whereby the conformation of the interfacial His 111 depends on the charge, and these differences in conformation may influence the solvation energy and the subsequent binding kinetics. Our results stress the complex relationship between environmental conditions and molecular binding properties.

Animals↗
PubMed1983

Conformational changes in benzodiazepine receptors induced by the antagonist Ro 15-1788.

The binding kinetics of [3H]Ro 15-1788, a selective benzodiazepine receptor antagonist, to synaptosomal membranes of rat cerebral cortices was studied. [3H]Ro 15-1788 binds with high affinity (dissociation constant, 0.53 nM) to a single class of binding sites (maximal binding capacity, 1.97 pmoles/mg of protein). Equilibrium binding was not affected by gamma-aminobutyric acid (GABA), NaCl, pentobarbital, or pretreatment of the membranes at 37 degrees. Association at 0 degrees was identical whether measured in the absence or presence of GABA or bicuculline methiodide or after preincubation of the membranes at 37 degrees. The association rate under pseudo-first order conditions was curvilinear and consisted of a fast component and a slow component. Dissociation at 0 degrees with 1 X 10(-5)M clonazepam was also curvilinear and could best be fitted by two linear exponential components. The dissociation rate was not altered by GABA, NaCl, pentobarbital, or pretreatment of membranes at 37 degrees. The dissociation rate was similar for 0.1, 1, and 10 nM [3H]Ro 15-1788. The ratio of slow to fast dissociation component for 10 nM [3H]Ro 15-1788 was larger than that for 0.1 and 1 nM [3H]Ro 15-1788. In contrast, the dissociation rate for 20 nM [3H]flunitrazepam ( [3H]FNP) was much greater than that for 2 nM [3H]FNP. Using ligand concentrations occupying the same fraction of receptors, the ratio of slow to fast dissociation components was invariably greater for [3H]Ro 15-1788 than that for [3H]FNP. The rate of dissociation for [3H]Ro 15-1788 was faster under pre-equilibrium conditions than under equilibrium conditions. These results, discussed in terms of the cyclic model of interaction between receptors and benzodiazepines, suggest that [3H]Ro 15-1788 is a powerful ligand in inducing conformational changes in the initial, more labile, binary complex. They also suggest that different conformational states deduced from studies of in vitro binding kinetics may not correspond to the distinct pharmacological actions of benzodiazepines. It is speculated that intrinsic activities of benzodiazepines probably are determined by the step beyond the complex formation and conformational changes suggested to occur by these studies of binding kinetics.

Animals↗
PubMed1975

Kinetics of binding of bovine trypsin-killikrein inhibitor (K unitz) in which the reactive-site peptide bond Lys-15--Ala-16 is cleaved, to alpha-chymotrypsin and beta-trypsin.

Equilibrium measurements of the binding of reactive-site-cleaved (modified) bovine trypsin-kallikrein inhibitor (Kunitz) to alpha-chymotrypsin and beta-trypsin show a stoichiometric 1:1 association with high binding constants. At least in the case of chymotrypsin much evidence is presented that the reaction with modified inhibitor leads to the same complex as the reaction with virgin inhibitor does. The association rate constant of modified inhibitor with chymotrypsin at pH 7, 22.5 degrees C is 15.8 M-1 S-1. This is about 2 x 10(4) times slower than the binding of virgin inhibitor to that enzyme. In the analogous reaction of modified inhibitor with beta-trypsin, however, the association rate constant (1.2 x 10(4) M-1 s-1 at pH 6.9, 22.5 degrees C) is of about the same order of magnitude as it is in the reaction of virgin inhibitor and trypsin. These and analogous phenomena observed in the reactions of virgin and modified soybean trypsin inhibitor (Kunitz) with alpha-chymotrypsin and beta-trypsin suggest that the specificity of both inhibitors to trypsin is strongly reflected in the association rate constants of the modified forms. The dissociation rate constants of the complexes of trypsin-kallikrein inhibitor with chymotrypsin or with trypsin towards the modified inhibitor are estimated to be unmeasurably slow (half-life times of 45 or 1.5 x 10(4) years, respectively).

Alanine↗