Binding of methotrexate to dihydrofolate reductase and its relation to protonation of the ligand

Stopped-flow spectrophotometry and stopped-flow fluorometry have been used to study the binding of methotrexate (MTX) and 3-deazamethotrexate (3-deazaMTX) to dihydrofolate reductase (DHFR) isoenzymes from Streptococcus faecium and from Lactobacillus casei. The absorbance change and fluorescence quenching that occur when MTX binds to DHFR isoenzyme II from S. faecium (SFDHFR II) are both biphasic and give similar apparent rate constants for both phases. The faster phase has an apparent rate constant that is dependent on MTX concentration and therefore corresponds to the initial binding reaction. From the concentration dependence it has been calculated that the association rate constant is 3.0 X 10(5) M-1 s-1 at 20 degrees C and pH 7.3, and the association constant (equilibrium constant) under these conditions is 5.8 X 10(5) M-1. By examination of the amplitude of the fast-phase absorbance change at various wavelengths, it has been determined that the absorbance change occurring in the fast phase is due to MTX protonation. Within the limits of the method it was thus not possible to detect a difference in the rates of binding and of protonation of MTX. The MTX association rate constant is pH dependent, decreasing 330-fold as the pH is decreased from 5.0 to 9.0. The data fit well to a curve generated by assuming a single ionization with a pKa of 6.0 and a pH-independent association rate constant 1000-fold greater for binding of protonated MTX to SFDHFR II than for binding of unprotonated MTX.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the formation and isomerization of methotrexate complexes of recombinant human dihydrofolate reductase

The kinetics of inhibitor binding to highly purified recombinant human dihydrofolate reductase (rHDHFR) have been examined. Methotrexate (MTX) binds rapidly (kon = 1.0 x 10(8) M-1 s-1) and tightly (koff/kon = 210 pM) to the preformed complex of rHDHFR with NADPH. The initial association reaction between rHDHFR.NADPH and MTX is followed by an isomerization of the resulting complex (kiso = 0.4 s-1) leading to a new conformer in which MTX is bound even more tightly (Ki = 3.4 pM). Similar results have been obtained with a major metabolite of MTX having four additional glutamate residues for which Ki = 1.4 pM. 7-HydroxyMTX, another major metabolite of MTX, is a weak inhibitor of rHDHFR (Ki = 8.9 nM), and a polyglutamate form of this metabolite is an equally weak inhibitor (Ki = 9.9 nM), so that the addition of glutamate residues to MTX or 7-hydroxyMTX has little effect on their binding. It follows that the significance of MTX polyglutamate formation relates to other roles such as increasing the cytotoxicity of MTX by prolonging intracellular retention of the drug. Another antifolate, trimethoprim, binds tightly to dihydrofolate reductases from bacterial sources, but weakly to rHDHFR in the ternary complex (KD = 0.5 microM). Although the association step is rapid (kon = 0.4 x 10(8) M-1 s-1), the dissociation rate is also rapid (koff = 15 s-1). Furthermore, there is no isomerization of the ternary complex of trimethoprim with rHDHFR, in contrast to the known isomerization of complexes of trimethoprim with bacterial dihydrofolate reductases.     

Role of aspartate 27 in the binding of methotrexate to dihydrofolate reductase from Escherichia coli

Dihydrofolate reductase from wild-type Escherichia coli (WT-ECDHFR) and from a mutant enzyme in which aspartate 27 is replaced by asparagine have been compared with respect to the binding of the inhibitor methotrexate (MTX). Although the Asp27----Asn substitution causes only small changes in the association rate constants (kon) for the formation of binary and ternary (with NADPH) complexes, the dissociation rate constants for these complexes (koff) are increased for the mutant enzyme by factors of about 5- and 100-fold, respectively, at pH 7.65. In binding experiments, the initial MTX binary and ternary complexes of the mutant enzyme were found to undergo relatively rapid isomerization (kobs approximately 17 and 145 s-1, respectively). Although such rapid isomerization of complexes of WT-ECDHFR could not be detected in binding experiments, evidence of a slow isomerization (k = 4 x 10(-3) s-1) of the ternary WT-ECDHFR.MTX.NADPH complex was obtained from progress of inhibition experiments. This slow isomerization increases binding of MTX to WT-ECDHFR only 2.4-fold (much less than previously estimated). From presently available data, we could not determine the contribution of the rapid isomerization of complexes to the binding of MTX to the mutant enzyme. The Asp27----Asn substitution increases the overall dissociation constant (KD) 9-fold for the binary complex and 85-fold for the ternary complex. When it is also taken into account that a proton ultimately derived from the solvent must be added to MTX bound to the WT enzyme, but not to MTX bound to the mutant enzyme, these increases in KD for the mutant enzyme correspond to decreases in binding energy for MTX of 3.9 and 5.2 kcal/mol at pH 7.65 for the binary and ternary complexes, respectively.     

Methotrexate, a high-affinity pseudosubstrate of dihydrofolate reductase.

Investigations have been made of the slow, tight-binding inhibition by methotrexate of the reaction catalyzed by dihydrofolate reductase from Streptococcus faecium A. Quantitative analysis has shown that progress curve data are in accord with a mechanism that involves the rapid formation of an enzyme-NADPH-methotrexate complex that subsequently undergoes a relatively slow, reversible isomerization reaction. From the Ki value for the dissociation of methotrexate from the E-NADPH-methotrexate complex (23 nM) and values of 5.1 and 0.013 min-1 for the forward and reverse rate constants of the isomerization reaction, the overall inhibition constant for methotrexate was calculated to be 58 pM. The formation of an enzyme-methotrexate complex was demonstrated by means of fluorescence quenching, and a value of 0.36 muM was determined for its dissociation constant. The same technique was used to determine dissociation constants for the reaction of methotrexate with the E-NADP and E-NADPH complexes. The results indicate that in the presence of either NADPH or NADP there is enhancement of the binding of methotrexate to the enzyme. It is proposed that methotrexate behaves as a pseudosubstrate for dihydrofolate reductase.     

Characteristics of Partially Purified Nerve Growth Factor Receptor

Receptors for the nerve growth factor protein (NGF) have been isolated from three cell types [embryonic chicken sensory neurons (dorsal root sensory ganglia; DRG), rat pheochromocytoma (PC12) and human neuroblastoma (LAN-1) cells] and have been shown to be similar with respect to equilibrium dissociation constants. The present results demonstrate that there are multiple molecular weight species for NGF receptors from DRG neurons and PC12 cells. NGF receptors can be isolated from DRG as four different molecular species of 228, 187, 125, and 112 kilodaltons, and PC12 cells as three molecular species of 203, 118, and 107 kilodaltons. The NGF receptors isolated from DRG show different pH-binding profiles for high- and low-affinity binding. High-affinity binding displays a bell-shaped pH profile with maximum binding between pH 7.0 and 7.9, whereas low-affinity binding is constant between pH 5.0 and 9.1, with a twofold greater binding at pH 3.6. At 22 degrees C, the association rate constant was found to be 9.5 +/- 1.0 X 10(6) M-1 s-1. Two dissociation rate constants were observed. The fast dissociating receptor has a dissociation rate constant of 3.0 +/- 1.5 X 10(-2) s-1, whereas the slow dissociating receptor constant was 2.4 +/- 1.0 X 10(-4) s-1. The equilibrium dissociation constants calculated from the ratio of dissociation to association rate constants are 2.5 X 109-11) M for the high-affinity receptor (type I) and 3.2 X 10(-9) M for the low-affinity receptor (type II). These values are the same as those determined by equilibrium experiments on the isolated receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of ternary complex formation between dihydrofolate reductase, coenzyme, and inhibitors

The kinetics of ligand binding to dihydrofolate reductase from Lactobacillus casei (MTX/R) to form the ternary enzyme-inhibitor-coenzyme complex have been investigated by the stopped-flow fluorescence technique. The fluorescence changes observed when coenzymes or inhibitors bind to the binary complex of the enzyme with the complementary ligand occur in a single fast phase. Under pseudo-first-order conditions the reaction traces could be fitted with precision to a single-exponential decay, and apparent bimolecular rate constants in the range 2 x 10(6) to 3 x 10(7) M-1s-1 have been measured assuming a bimolecular-unimolecular model. The kinetic constants obtained suggest that prior binding of an inhibitor to the enzyme may, to a minor extent, interfere with coenzyme binding but the rates of inhibitor binding seem to be unaffected by the presence of a bound coenzyme. Dissociation rate constants appear to be less than 1 s-1 which suggests that both coenzymes and inhibitors are tightly bound in the ternary complex. An investigation of the effects of pH on the kinetics of ternary complex formation indicated the involvement of ionizable groups in ligand binding, but this shows some ligand dependence. The rates of ligand bindings to form the ternary complex are fairly high, but it is unlikely that these associations are diffusion controlled because their measured activation energies of 7.8-14.5 kcal mol-1 are higher than expected from reactions whose rates are limited by diffusion in aqeous solution.     

Transient state kinetics of the reactions of isobutyraldehyde with compounds I and II of horseradish peroxidase

Elementary reactions have been studied quantitatively in the complex overall process catalyzed by horseradish peroxidase whereby isobutyraldehyde and molecular oxygen react to form triplet state acetone and formic acid. The rate constant for the reaction of the enol form of isobutyraldehyde with compound I of peroxidase is (8 +/- 1) X 10(6) M-1 s-1 and with compound II (1.3 +/- 0.3) X 10(6) M-1 s-1. Neither the enolate anion nor the keto form is reactive. The reactivity of enols with peroxidase parallels that of unionized phenols and a common mechanism is proposed. The overall catalyzed reaction of isobutyraldehyde and oxygen consists of an initial burst followed by a steady state phase. The burst is caused by the following sequence: 1) an initial high yield of compound I is formed from reaction of native enzyme with the autoxidation product of isobutyraldehyde, a peracid and 2) compound I rapidly depletes the equilibrium pool of enol which is present. After this burst a steady state phase is observed in which the rate-limiting step is the conversion of the keto to the enol form of the aldehyde catalyzed by phosphate buffer. The rate constant for the keto form reacting with phosphate is (8.7 +/- 0.6) X 10(-5) M-1 s-1. All constants were measured in dilute aqueous ethanol at 35 degrees C, pH 7.4, and ionic strength 0.67 M. Both the initial burst of light and the steady state emission from triplet acetone can be observed with the naked eye. Since the magnitude of the burst is a measure of the equilibrium amount of enol, the keto-enol equilibrium constant is readily calculated and hence also the rate constant for conversion of enol to keto. The keto-enol equilibrium constant is unaffected by phosphate which therefore acts as a true catalyst.     

Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei.

Dihydrofolate reductase has been purified from a methotrexate-resistant strain of Lactobacillus casei NCB 6375. By careful attention to growth conditions, up to 2.5 g of enzyme is obtained from a 400 litre culture. The purification procedure, involving poly-ethyleneimine treatment, DEAE-cellulose chromatography and affinity chromatography on methotrexate-aminohexyl-Sepharose, operates on the gram scale, with overall yields of 50-60%. Elution of the affinity column by reverse (upward) flow was used, as it led to recovery of the enzyme in a much smaller volume. The enzyme obtained appears to be more than 98% pure, as judged by gel electrophoresis, isoelectric focusing, and gel filtration. It has a mol.wt. of approx. 17900 and a turnover number of 4s-1 (50mM-triethanolamine/400mM-KCl, pH 7.2, 25 degrees C) with dihydrofolate and NADPH as substrates. The turnover number for folate is 0.02s-1. Michaelis constants for a variety of substrates have been measured by using a new fluorimetric assay (0.36 muM-dihydrofolate; 0.78 muM-NADPH), and binding constants determined by using the quenching of protein fluorescence (dihydrofolate, 2.25 X 10(6)M-1; NADPH, greater than 10(8)M-1). The pH/activity profile shows a single maximum at pH 7.3; at this pH, marked activation by 0.5M-NaCl is observed.     

Kinetic and equilibrium studies on steroid interaction with human corticosteroid-binding globulin

Kinetic and equilibrium studies on the interaction of steroids with human corticosteroid-binding globulin (CBG, transcortin) were performed with pH, temperature, and steroid structure as variables. Dissociation rate constants were determined fluorometrically; the values for cortisol, corticosterone, deoxycorticosterone, and progesterone are 0.031, 0.047, 0.10, and 0.16 s-1, respectively, at 20 degrees C, pH 7.4. The pH dependence of the dissociation rate constant for the corticosterone complex below pH 10.5 at 20 degrees C is given by koff = 0.043 (1 + [H+]/10(-6.50)) s-1; above pH 11, koff = 0.030 (1 + 10(-12.15/[H+] s-1. A temperature-dependence study of koff for the cortisol and progesterone complexes gave values of 0.0028 s-1 and 0.012 s-1 at 4 degrees C, respectively, and 0.88 s-1 and 4.5 s-1 at 37 degrees C, with progesterone dissociating about four to five times faster over the entire temperature range. The affinity constants, determined by equilibrium dialysis, for the binding of cortisol, corticosterone, and progesterone at 4 degrees C were 7.9, 7.2, and 7.0 X 10(8) M-1; values of 0.40 and 0.26 X 10(8) M-1 were determined at 37 degrees C for cortisol and progesterone. The close similarity of the affinity constants of the three steroids combined with differing dissociation rates implies that the association rate changes with steroid structure, in contrast to our earlier findings with progesterone-binding globulin.     

Kinetic studies on the interaction of eglin c with human leukocyte elastase and cathepsin G

We have investigated the inhibition of human leukocyte elastase and cathepsin G by recombinant Eglin c under near physiological conditions. The association rate constants k on of Eglin c for elastase and cathepsin G were 1.3 X 10(7) M-1 s-1 and 2 X 10(6) M-1 s-1, respectively. Under identical conditions, the k on for the association of human plasma alpha 1-proteinase inhibitor with the two leukocproteinases were 2.4 X 10(7) M-1 s-1 and 10(6) M-1 s-1, respectively. The consistency of these data could be verified using a set of competition experiments. The elastase-Eglin c interaction was studied in greater detail. The dissociation rate constant k off was determined by trapping of free elastase from an equilibrium mixture of elastase and Eglin c with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The rate of dissociation was very low (k off = 3.5 X 10(-5) s-1). The calculated equilibrium dissociation constant of the complex, Ki(calc) = k off/k on, was found to be 2.7 X 10(-12) M. Ki was also measured by adding elastase to mixtures of Eglin c and substrate and determining the steady-state rates of substrate hydrolysis. The Ki determined from these experiments (7.5 X 10(-11) M) was significantly higher than Ki(calc). This discrepancy might be explained by assuming that the interaction of Eglin c with elastase involves two steps: a fast binding reaction followed by a slow isomerization step. From the above kinetic constants it may be inferred that at a therapeutic concentration of 5 X 10(-7) M, Eglin c will inhibit leukocyte elastase in one second and will bind this enzyme in a "pseudo-irreversible" manner.     

Rate constants for a mechanism including intermediates in the interconversion of ternary complexes by horse liver alcohol dehydrogenase

Transient kinetic data for partial reactions of alcohol dehydrogenase and simulations of progress curves have led to estimates of rate constants for the following mechanism, at pH 8.0 and 25 degrees C: E in equilibrium E-NAD+ in equilibrium *E-NAD+ in equilibrium E-NAD(+)-RCH2OH in equilibrium E-NAD+-RCH2O- in equilibrium *E-NADH-RCHO in equilibrium E-NADH-RCHO in equilibrium E-NADH in equilibrium E. Previous results show that the E-NAD+ complex isomerizes with a forward rate constant of 620 s-1 [Sekhar, V. C., & Plapp, B. V. (1988) Biochemistry 27, 5082-5088]. The enzyme-NAD(+)-alcohol complex has a pK value of 7.2 and loses a proton rapidly (greater than 1000 s-1). The transient oxidation of ethanol is 2-fold faster in D2O, and proton inventory results suggest that the transition state has a charge of -0.3 on the substrate oxygen. Rate constants for hydride ion transfer in the forward or reverse reactions were similar for short-chain aliphatic substrates (400-600 s-1). A small deuterium isotope effect for transient oxidation of longer chain alcohols is apparently due to the isomerization of the E-NAD+ complex. The transient reduction of aliphatic aldehydes showed no primary deuterium isotope effect; thus, an isomerization of the E-NADH-aldehyde complex is postulated, as isomerization of the E-NADH complex was too fast to be detected. The estimated microscopic rate constants show that the observed transient reactions are controlled by multiple steps.     

Kinetics of the reaction between testosterone and antitestosterone antiserum

In order to characterize from a kinetic viewpoint the antibody population mainly involved in the binding of testosterone by its homologous antiserum, the kinetics of the association reaction between [1,2,6,7-3H]-testosterone and rabbit antiserum anti-testosterone-3-(O-carboxymethyl)oxime-bovine serum albumin (Ab R2603-1) was followed at pH 7.4 and at constant ionic strength, at temperatures ranging from 2 degrees C to 37 degrees C and at concentration near to work conditions for testosterone radioimmunoassay; dextran coated charcoal suspension was used for the bound/free separation. In the examined concentration range, the observed kinetics trends can be explained by assuming the existence of two classes of antibody binding sites, Ab1 and Ab2. The kinetics of the dissociation reaction of the testosterone-antibody complex was also followed after the addition of a large excess of unlabeled testosterone. At 22.0 degrees C, association and dissociation rate constants are 2.1.10(7) s-1M-1 and 3.7.10(-3) s-1, respectively, for the Ab1 class of antibody binding sites, and 3.6.10(6) s-1M-1 and 7.0.10(-4) s-1 for the Ab2 class. Equilibrium constants obtained from kinetic data were very similar for both classes of antibody binding sites and in good agreement with the equilibrium values obtained from linear Scatchard plot. The order of magnitude of the second order rate constants and the high activation enthalpy for the forward and reverse reaction suggest a mechanism more complex than a simple second order.     

The reaction between cytochrome c1 and cytochrome c

The kinetics of electron transfer between the isolated enzymes of cytochrome c1 and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c1 and ferricytochrome c is fast; the second-order rate constant (k1) is 3.0 . 10(7) M-1 . s-1 at low ionic strength (I = 223 mM, 10 degrees C). The value of this rate constant decreases to 1.8 . 10(5) M-1 . s-1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c1 and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c1 and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c1 and ferricytochrome c and the reverse reaction are k1 = 2.4 . 10(5) M-1 . s-1 and k-1 = 3.3 . 10(5) M-1 . s-1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10 degrees C). The 'equilibrium' constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c2+1 + cytochrome c3+ in equilibrium or formed from cytochrome c3+1 + cytochrome c2+.     

Characterization of the oxycomplex of lignin peroxidases from Phanerochaete chrysosporium: equilibrium and kinetics studies

The oxycomplexes (compound III, oxyperoxidase) of two lignin peroxidase isozymes, H1 (pI = 4.7) and H8 (pI = 3.5), were characterized in the present study. After generation of the ferroperoxidase by photochemical reduction with deazoflavin in the presence of EDTA, the oxycomplex is formed by mixing ferroperoxidase with O2. The oxycomplex of isozyme H8 is very stable, with an autoxidation rate at 25 degrees C too slow to measure at pH 3.5 or 7.0. In contrast, the oxycomplex of isozyme H1 has a half-life of 52 min at pH 4.5 and 29 min at pH 7.5 at 25 degrees C. The decay of isozyme H1 oxycomplex follows a single exponential. The half-lives of lignin peroxidase oxycomplexes are much longer than those observed with other peroxidases. The binding of O2 to ferroperoxidase to form the oxycomplex was studied by stopped-flow methods. At 20 degrees C, the second-order rate constants for O2 binding are 2.3 X 10(5) and 8.9 X 10(5) M-1 s-1 for isozyme H1 and 6.2 X 10(4) and 3.5 X 10(5) M-1 s-1 for isozyme H8 at pH 3.6 and pH 6.8, respectively. The dissociation rate constants for the oxycomplex of isozyme H1 (3.8 Z 10(-3) s-1) and isozyme H8 (1.0 X 10(-3) s-1) were measured at pH 3.6 by CO trapping. Thus, the equilibrium constants (K, calculated from kon/koff) for both isozymes H1 (7.0 X 10(7) M-1) and H8 (6.2 X 10(7) M-1) are higher than that of myoglobin (1.9 Z 10(6) M-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

The kinetics of Schiff-base formation during reconstitution of D-serine apodehydratase from Escherichia coli with pyridoxal 5'-phosphate.

Schiff base formation during reconstitution of D-serine dehydratase (Escherichia coli) from its apoenzyme and pyridoxal 5'-phosphate (pyridoxal-P) has been studied by rapid kinetic techniques using absorbance changes at 436 nm. Three distinct reaction phases have been observed. The first is a very rapid change during which pyridoxal-P is initially bound to the apoenzyme. This step has an equilibrium constant of 1500 M-1 and a forward reaction rate of the order of 2.6 x 10(6) M-1 s-1. The second phase shows a first-order rate constant with a value dependent on pyridoxal-P and corresponds to a first-order step with a forward rate constant of 3.04 s-1 interacting with the initial equilibrium. The final phase is a slow first-order reaction, the rate constant of which is approximately 0.01 s-1 and is independent of pyridoxal-P concentration. The active pyridoxal species has been shown to be the free pyridoxal-P as opposed to hemiacetal or hemimercaptal forms.     

Kinetics of binding of oligosaccharides to a homogeneous pneumococcal antibody: dependence on antigen chain length suggests a labile intermediate complex.

Temperature-jump experiments were performed with di-, tetra-, and hexasaccharides derived from type III pneumococcal polysaccharide using a homogeneous corresponding antibody IgG 45-394. A decrease in stability of the oligosaccharide-antibody complexes with decreasing chain length was observed and entirely reflected in the decrease of the association rate constants which were 1.7 X 10(4) M-1 s-1 for the di-, 3.7 X 10(5) M-1 s-1 for the tetra-, and 1.1 X 10(6) M-1 s-1 for the hexasaccharide at 23 degrees C. The dissociation rate constants for all oligomers were about 12 s-1. This marked chain-length dependence of the association rate constants as well as their low values are unexpected for a single binding step. A mechanism is proposed which consists of a fast formation of a labile oligosaccharide-antibody precomplex followed by a slow isomerization step which is induced by the oligosaccharide ligands but which is chain-length independent.     

Kinetics of phloretin binding to phosphatidylcholine vesicle membranes

The submillisecond kinetics for phloretin binding to unilamellar phosphatidylcholine (PC) vesicles was investigated using the temperature-jump technique. Spectrophotometric studies of the equilibrium binding performed at 328 nm demonstrated that phloretin binds to a single set of independent, equivalent sites on the vesicle with a dissociation constant of 8.0 microM and a lipid/site ratio of 4.0. The temperature of the phloretin-vesicle solution was jumped by 4 degrees C within 4 microseconds producing a monoexponential, concentration-dependent relaxation process with time constants in the 30--200-microseconds time range. An analysis of the concentration dependence of relaxation time constants at pH 7.30 and 24 degrees C yielded a binding rate constant of 2.7 X 10(8) M-1 s-1 and an unbinding constant of 2,900 s-1; approximately 66 percent of total binding sites are exposed at the outer vesicle surface. The value of the binding rate constant and three additional observations suggest that the binding kinetics are diffusion limited. The phloretin analogue, naringenin, which has a diffusion coefficient similar to phloretin yet a dissociation constant equal to 24 microM, bound to PC vesicle with the same rate constant as phloretin did. In addition, the phloretin-PC system was studied in buffers made one to six times more viscous than water by addition of sucrose or glycerol to the differ. The equilibrium affinity for phloretin binding to PC vesicles is independent of viscosity, yet the binding rate constant decreases with the expected dependence (kappa binding alpha 1/viscosity) for diffusion-limited processes. Thus, the binding rate constant is not altered by differences in binding affinity, yet depends upon the diffusion coefficient in buffer. Finally, studies of the pH dependence of the binding rate constant showed a dependence (kappa binding alpha [1 + 10pH-pK]) consistent with the diffusion-limited binding of a weak acid.     

A kinetic study of cyclic adenosine 3':5'-monophosphate binding and mode of activation of protein kinase from Drosophila melanogaster embryos.

Cyclic AMP-dependent protein kinase and its regulatory subunit were isolated from Drosophila melanogaster embryos. The profiles of cyclic AMP binding by these proteins were significantly different. In order to explain such a difference and to find the mode of enzyme activation by cyclic AMP, a kinetic study of cyclic AMP binding was carried out. First, the association rate constant k1 and dissociation rate constant k-1 in the cyclic AMP-regulatory subunit interaction at 0 degrees C were estimated to be 2.3 X 10(6)M-1s-1 and 1.1 X 10(-3)s-1, respectively. Secondly, the three possible modes of enzyme activation by cyclic AMP were mathematically considered and could be described by a unique formula: r=APt + BQt (A + B=1) in which the parameters A, B, P, and Q are equivalent to rate constants in the sense that the rate constants are simply expressed by these parameters. Thirdly, the values of the parameters and subsequently the values of rate constants involved in the possible mechanisms were evaluated using a curve-fitting technique and compared with experimental observation. It was then found that the following mechanism was the only one which fitted the experimental observations. Namely, RC + L k3 equilibrium k-3 LRC k4 equilibrium k-4 RL + C where R, C, and L represent the regulatory and catalytic subunits and cyclic AMP as a ligand. Thus, our results indicate that in the presence of cyclic AMP the active enzyme (C) is released from a ternary intermediate which is the primary product of the cyclic AMP-holoenzyme interaction. The estimated values of the rate constants are: k3=3.5 X 10(6)M-1s-1;k-3=7.3 X 10(-1)s-1;and k4=3.8 X 10(-2)s. These estimates indicate that the reaction LRC leads to RL + C is relatively slow and limits the rate of the overall reaction. By comparing k-3 and k4, it is apparent that a large part of newly formed ternary intermediate reverts to the holoenzyme.     

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)     

Rate constants and equilibrium constants for binding of the gelsolin-actin complex to the barbed ends of actin filaments in the presence and absence of calcium

The equilibrium constant for binding of the gelsolin-actin complex to the barbed ends of actin filaments was measured by the depolymerizing effect of the gelsolin-actin complex on actin filaments. When the gelsolin-actin complex blocks monomer consumption at the lengthening barbed ends of treadmilling actin filaments, monomers continue to be produced at the shortening pointed ends until a new steady state is reached in which monomer production at the pointed ends is balanced by monomer consumption at the uncapped barbed ends. By using this effect the equilibrium constant for binding was determined to be about 1.5 X 10(10) M-1 in excess EGTA over total calcium (experimental conditions: 1 mM MgCl2, 100 mM KCl, pH 7.5, 37 degrees C). In the presence of Ca2+ the equilibrium constant was found to be in the range of or above 10(11) M-1. The rate constant of binding of the gelsolin-actin complex to the barbed ends was measured by inhibition of elongation of actin filaments. Nucleation of new filaments by the gelsolin-actin complex towards the pointed ends was prevented by keeping the monomer concentration below the critical monomer concentration of the pointed ends where the barbed ends of treadmilling actin filaments elongate and the pointed ends shorten. The gelsolin-actin complex was found to bind fourfold faster to the barbed ends in the presence of Ca2+ (10 X 10(6) M-1 s-1) than in excess EGTA (2.5 X 10(6) M-1 s-1). Dissociation of the gelsolin-actin complex from the barbed ends can be calculated to be rather slow. In excess EGTA the rate constant of dissociation is about 1.7 X 10(-4) s-1. In the presence of Ca2+ this dissociation rate constant is in the range of or below 10(-4) s-1.