A kinetic study of -acetohydroxy acid isomeroreductase from Salmonella typhimurium

The kinetic mechanism of α-acetohydroxy acid isomeroreductase from Salmonella typhimurium has been investigated by initial velocity kinetic and product inhibition studies. The results of the initial velocity studies are consistent with a sequential reaction. The product inhibition studies suggest an ordered reaction with NADPH and the acetohydroxy acid adding in that order, and dihydroxy acid release before NADP release.NADPH binding has been studied both by fluorimetric techniques and difference spectroscopy. From these investigations it has been calculated that 4 moles of NADPH bind per mole of enzyme; the first molecule of NADPH binds with a dissociation constant of 1.7 × 10^?6m, the subsequent 3 moles of NADPH bind with a constant of 6 × 10^?6m. Biphasic kinetics have been demonstrated at a wide range of NADPH concentrations. The occurrence of biphasic kinetics and two separate binding constants are discussed in terms of negative cooperativity.     

The reaction of cytochalasin a with sulfhydryl groups

The reaction of cytochalasin A with sulfhydryl groups was examined. Cysteine and glutathione reacted readily with cytochalasin A at pH 7.0, 20°C, following second-order kinetics with rate constants of 7,600 M^?1 sec^?1 and 870 × 10³ M^?1 sec^?1. No reaction of cytochalasin B could be demonstrated under the same conditions. The reaction of cytochalasin A with the amino group of glycine ethyl ester had a second-order rate constant of 0.02 M^?1 sec^?1. Cytochalasin A did not react with sufhydryl groups of native ovalbumin or lactic dehydrogenase but reacted with an least 2 and 12 groups respectively when the proteins were denatured in 0.1% SDS. The reactivity of cytochalasin A with sulfhydryl groups is attributable to the α,β-unsaturated ketone groups it contains.     

Kinetics of complex formation between fluorescein mercuric acetate and DNA

The kinetics of complex formation between fluorescein mercuric acetate and heat-denatured DNA were studied by measuring the fluorescence quenching of this reagent. This quenching process involved no immeasurably rapid phase and it was shown that this reaction follows simple second-order kinetics. The rate constant at 25°C was estimated to be 2.9 × 10⁴M^?1 sec^?1 for calf-thymus DNA (42% G + C) and 1.1 × 10⁴M^?1 sec^?1 for Micrococcus lysodeikticus DNA (72% G + C). Activation parameters for this reaction were calculated from the temperature dependence of the reaction rate, and the activation entropy was found to be highly negative (?27.5 cal/mol deg for calf-thymus DNA and ?25.5 cal/mol deg for M. lysodeikticus DNA). The binding of fluorescein mercuric acetate to native DNA, which requires the opening of the double-helical structure, was also followed by measuring the absorbance change of this reagent. There was a lag phase in this binding process, and the enthalpy change for the opening step corresponded roughly to that for the opening of one base pair. These findings are discussed in relation to the results of a similar study with formaldehyde.     

Enzymatic studies on the interaction of myosin and heavy meromyosin with 1,N 6 -ethenoadenosine triphosphate ( ATP), a fluorescent analog of ATP

The fluorescent analog of adenosine triphosphate (ATP)¹ 1,N⁶-ethenoadenosine triphosphate, (εATP), has been utilized as a substitute for ATP in the myosin and heavy meromyosin ATPase systems. For myosin, the analog εATP replaced ATP with a somewhat larger K_m (2.6 × 10^?4 mole ?^?1 for εATP as opposed to 8.8 × 10^?5 mole ?^?1 for ATP), indicating that the apparent affinity of the enzyme for εATP is less than for ATP. Perhaps of more interest, further comparison yielded a V_max for εATP about two and one half times the value for ATP (20 μmole PO₄ sec^?1 g protein^?1 as opposed to 8.1 μmole sec^?1 g protein^?1). Results for the HMM-εATPase system were similar, yielding a K_m value of 1.47 × 10^?4 mole ?^?1 and a V_max of 54.2 μmole PO₄ sec^?1 g protein^?1, as opposed to corresponding K_m and V_max values of 1.23 × 10^?4 mole ?^?1 and 20.4 μmole PO₄ sec^?1 g protein^?1, respectively for the HMM-ATP interaction. The pH dependence of εATPase for both systems was comparable to ATP, suggesting a similarity in the mechanism of hydrolysis of the two nucleotides. Activation of εATPase by Ca²⁺ in the presence of 0.5 M KCl was comparable to ATPase for both systems, but inhibition by Mg²⁺ seemed to be more effective for εATPase. These results indicate that εATP is an excellent substitute for ATP in the myosin and heavy meromyosin systems and because of its insertion into the active site of these muscle proteins, it promises to be a very useful probe for conformation studies at this level.     

Kinetics of the acid hydrolysis of heparin from its Cu (II) complex and its relation to biological activity

The acid-catalyzed hydrolysis of heparin from Cu(II) complex was studied as a function of time and temperature. Four independent calculations showed that the hydrolysis, during the 5-hr period examined, obeys the first-order kinetic law. Specific rate constants, calculated at 50°C, 57°C, 65°C, 71°C, and 80°C, were 3.3 × 10^?5 sec^?1, 6.5 × 10^?5 sec^?1, 10.4 × 10^?5 sec^?1, 15.1 × 10^?5 sec^?1, and 26.6 × 10^?5 sec^?1, respectively. Arrhenius plots of the data yielded 14.7 kcal as the energy of activation. An independent run of the self-hydrolysis of heparin at 57°C also obeyed first-order kinetics and its specific rate constant of 6.4 × 10^?5 sec^?1 is in excellent agreement with that of the hydrolysis of Cu(II)-heparin at 57°C. The anticoagulant activity of heparin and of the Cu(II)-heparin are not appreciably different. Further, the inactivation of heparin closely parallels Cu(II) release from the Cu(II) complex which in turn parallels desulfation.     

Hydrolysis of p-nitrotrifluoroacetanilide catalyzed by water and imidazole

The hydrolyses of p-nitrotrifluoroacetanilide catalyzed by water and imidazole were examined at 70°C. The pH-rate constant profile of the hydrolysis in H₂O was examined in the pH range 0.0–11.4. The hydrolysis was independent of pH in the region from pH 1.0 to 4.5, presumably a water-catalyzed reaction. The rate constant and the D₂O solvent isotope effect for this reaction were 1.0 × 10^?4 sec^?1 and 3.7, respectively. Both natural imidazole and imidazolium cation catalyzed hydrolysis. The rate constant of the hydrolysis catalyzed by neutral imidazole was determined to be 5.4 × 10^?3M^?1 sec^?1 and the D₂O solvent isotope effect was 1.8.     

A facile synthesis of ceramides.

Lateral diffusion of phosphatide molecules in liquid crystalline bilayers has been analysed as a case of co-operative lattice diffusion. The potential energy of interaction between two molecules is assumed to arise from Van der Waals interactions of the hydrocarbon chains, and to have the form suggested by Salem [6]. From the observed values of the self-diffusion constant (of the order of 10^?8 cm² sec^?1) the depth of the potential “well” for two molecules at the equilibrium separation was estimated to have a lower limit of 1.95 kcal per mole, and the energy barrier to lateral motion was estimated to have an upper limit of 7.21 kcal per mole.     

Reactions of oxygen radical species with methional: a pulse radiolysis study.

The reaction of hydroxyl radicals (^?OH) and superoxide anions ($\text{O}{}_2{}^{\mathrm{?}}$) with methional were investigated by pulse-radiolytic methods. The second-order rate constant for the attack of OH was determined at 8.2×10⁹ M^?1 sec^?1. In the case of $\text{O}{}_2{}^{\mathrm{?}}$ a slow first-order decay rate of 5.2×10³ sec^?1 suggests a far less efficient reaction. The transient species were identified by comparison with published results of pulse radiolysis and EPR spectroscopy of model compounds. The mechanism for the oxidation of methional by OH was found to be more complex than a simple fragmentation reaction.     

Kinetics of formation of ferrioxamine B

Stopped-flow kinetic studies of the formation of ferrioxamine B were performed. Formation of the complex follows the rate law

where K_a is the acid dissociation constant of the iron(III) aquo species in 0.1 M formate buffer. At 25°C k₁ = 3.94 × 10²M^?1 sec^?1, k₂K_a = 1.18 × 10^?1 sec^?1, k₃ = 3.6 × 10^?1 sec^?1. Activation parameters for k₁ are ΔH^≠ = 11.7 kcal mole^?1 and ΔS^≠ = ?8 cal K^?1 mole^?1. An associative mechanism is proposed. Attachment of the first chelate ring is the slow step and favorably positions the second chelate ring for attachment. Coordination of two chelate rings favorably positions the third chelate ring for attachment. These results are compared to kinetics of formation of model complexes and to a previous study of the formation of ferrioxamine B in which attachment of the third chelate ring was proposed as the slow step     

The nature and reactivity of the ‘essential’ thiol in rabbit muscle creatine kinase III (EC 2.7.3.2)

Rabbit muscle creatine kinase III (EC 2.7.3.2) can be reacted with 2-chloromercuri-4-nitrophenol and this results in the incorporation of two moles of mercurial per mole of enzyme subunit in a biphasic reaction. The second-order rate constant for the slow reaction is 475 ± 42 M^?1 s^?1. S-Carboxamidomethyl-creatine kinase reacts with a single mole of mercurial per mole of subunit. The rate constant, 466 ± 57 M^?1 s^?1, is almost identical to that for the slow reaction of the native enzyme. The reaction between 3-carboxy-4-nitrophenylthio-creatine kinase and 2-chloromercuri-4-nitrophenol has a second-order rate constant of 449 ± 56 M^?1 s^?1. The results may be explained if the mercurial reacts very rapidly with that cysteine residue which reacts independently with iodoacetamide or 5,5′-dithiobis(2-nitrobenzoic acid). However, 2-chloromercuri-4-nitrophenol also reacts more slowly with a second cysteine residue. Definition of the essentiality of thiol groups in enzymes by reaction with labile ligands, here represented by organomercurials, clearly must be approached with caution.     

Hydrogen-deuterium exchange of the tryptophan residues in bovine α-lactalbumin

Effects of deuteration on the Raman spectrum of a tryptophan residue have been examined. The 1386 cm^?1 line of deuterated tryptophan residue has been found to be useful for tracing the hydrogen-deuterium exchange reaction of this residue in a protein. An examination on bovine α-lactalbumin at pH 6.4 and at 20°C indicates that two of the four tryptophan residues exchange with a rate constant much greater than 9 × 10^?4 sec^?1, while the other two exchange with a rate constant of 4 × 10^?5 sec^?1. The latter two have been assigned to Trp 28 and Trp 108 of this protein. The kinetics of hydrogen-deuterium exchange reaction of completely “free” tryptophan residue have been examined by a proton magnetic resonance study on tryptophan itself. By taking the result of this examination into account, the chance of exposure to the solvent for Trp 28 or Trp 108 has been estimated to be 3 × 10^?6 at pH 6.4 and at 20°C.     

Nonenzymatic reduction of nitrosobenzene to phenylhydroxylamine by NAD(P)H

The nonenzymatic reduction of nitrosobenzene by NADPH and NADH in aqueous buffer solution at 25°C is described. Both reactants quantitatively convert nitrosobenzene to phenylhydroxylamine. Rate constants for reduction (k_r) were determined spectrophotometrically and found to be identical at pH 5.7 and 7.4 and independent of buffer concentration. The values of k_NADH (124–149 M^?1 sec^?1) and k_NADPH (131–170 M^?1 sec^?1) are essentially identical. The reaction is not subject to general catalysis or specific salt effects. The oxidation of phenylhydroxylamine by NAD(P) to nitrosobenzene is only stimulated by a factor of 1.2 over oxidation in its absence (when the ratio of NADP: phenylhydroxylamine was 8:1).     

Application of band centrifugation to the study of the assembly of alfalfa mosaic virus

A band-centrifugation method for the analysis of an assembly reaction of a simple virus from its RNA and protein is described. The experiment was carried out by sedimenting a band of viral RNA through a solution of depolymerized coat protein. The resulting radial distribution of the reaction products, followed as a function of time, was analyzed by a computer simulation of the series of reaction. This method is based on a numerical solution of the continuity equation for the sedimentation–diffusion process [Claverie, J.-M., Dreux, H. & Cohen, R. (1975) Biopolymers 14 , 1685–1700; Cohen, R. & Claverie, J.-M. (1975) Biopolymers 14 , 1701–1716]. A numerical method for the simulation of the chemical reaction is derived. From the simulated reaction series, equilibrium constants emerge for the successive addition of protein subunits to the growing nucleoprotein particle. The method is applied to the assembly of alfalfa mosaic virus. If the reaction between RNA and protein is carried out in 0.32M CsCl, pH 7.0, two stages during particle growth are resolved, each characterized by an equilibrium constant K. The determined values for K range from 5 × 10⁵ to 3 × 10⁶ l. mol^?1. The existence of these two stages may have a structural implication in the assembly, as they likely represent an elongation and a termination stage. If the reaction is carried out under more favorable conditions (0.25M CsCl, pH 7.0), a kinetic constant of at least 10⁵ l. mol^?1 sec^?1 is derived for each reaction step. Under these conditions the assembly appears to be completed within 1 min, which is too fast to detect distinct stages by band sedimentation.     

Anomeric specificity of fructosediphosphate aldolase E.C.4.1.2.13 from rabbit muscle

Fructosediphosphate aldolase from rabbit muscle is shown to accept β-D-fructofuranose-1,6-diphosphate as substrate, whereas α-D-fructofuranose-1,6-diphosphate can only be cleaved by the enzyme after a spontaneous change of configuration. The first order velocity constant of the spontaneous reaction was computed to be 0.55 sec^?1 (at 25° C, pH 7.6). The equilibrium mixture of D-fructose-1,6-diphosphate was computed to 26% α- and 74% β-D-fructofuranose-1,6-diphosphate.     

The role of copper binding in the conformation of stellacyanin

The binding of Cu²⁺ to apostellacyanin occurs in two steps. The first step consists of a fast equilibrium reaction involving binding of copper to the protein in a non-native, though specific way, as shown by electron paramagnetic resonance measurements. All the spectroscopic properties of native stellacyanin are recovered in a slower monomolecular process (k = 7.5 × 10^?3 sec^?1 at 25 °C) characterized by high activation energy (ΔH_a = 22 kcal mole^?1) and low activation entropy (ΔS_a = 3.0 cal deg^?1 mole^?1). The second step parallels a conformational change of the copper-bound protein molecule. A large difference of the tyrosyl residues pKs is found between holo- and apostellacyanin. In the latter the tyrosyl residues appear to be more exposed to solvent perturbations. Ammonia or monovalent anions such as N₃^?, SCN^?, and Cl^? have a catalytic effect on the second step of the reaction, roughly proportional to their first binding constant to aqueous copper. It is suggested that they may compete for a non-native bond of the copper to the protein, thus rendering the conformational change easier.The effect of Ag³ and Hg²⁺ on the recombination reaction with copper is discussed in terms of conformation of the metal-bound protein.     

Oxidation of ascorbic acid with superoxide anion generated by the xanthine-xanthine oxidase system.

Ascorbic acid was found to be oxidized by O₂^$\text{?}$ which was generated by the xanthine-xanthine oxidase system. From a kinetic analysis of the inhibition of this reaction by superoxide dismutase, the second-order rate constant for the reaction between ascorbic acid and O₂^? at pH 7.4 was estimated to be 2.7 × 10⁵ M^?1 sec^?1. A function of ascorbic acid as a defense against O₂^? is presented.     

Structural and physicochemical analysis of the reaction between the anti-lysozyme antibody D1.3 and the anti-idiotopic antibodies E225 and E5.2

The reaction between the mouse (BALB/c) anti-idiotiopic monoclonal antibodies E225 and E5.2 and idiotopes on the (BALB/c) anti-lysozyme monoclonal antibody D1.3 has been characterized by titration calorimetry, by equilibrium sedimentation and by the determination of binding association and dissociation rates. The reaction between E5.2 and D1.3 is driven by a large negative enthalpy and its rate and equilibrium association constants are comparable to those observed in other antigen–antibody reactions. In contrast, the reaction between E225 and D1.3 is entropically driven and characterized by slow association kinetic (1 × 10³ M^?1 sec^?1) and a resulting low equilibrium constant (K_a = 2 × 10⁵M ^?1). A correlation of these properties with the three-dimensional structure of the Fab225-FabD1.3 complex, previously determined by X-ray diffraction methods to 2.5 Å resolution, indicates that conformational changes of several D1.3 contacting residues, located in its complementarity determining regions, may explain these features of the reaction.     

Thermodynamic and kinetic parameters of an oligonucleotide hairpin helix

The thermodynamics of the hairpin helix-single strand transition of A₆C₆U₆ has been analyzed by a staggering zipper model with consideration of single strand stacking. This analysis yields an enthalpy change of +11 kcal/mole for the formation of a first, isolated base pair. The stability constant of a first (intramolecular) base pair in A₆C₆U₆ is around 2 × 1O^?5 at 25°C, whereas a first (intermoleciilar) base pair in an A₆ · U₆ helix is characterised by a stability constant of about 4 × 10^?3M^?1 (25°C, extrapolated from A_n · V_n oligomer measurements). These data indicate a destabilizing effect of the C₆ loop.The rate constant of hairpin helix formation is 2 to 3 × 10⁴ sec^?1 associated with an activation enthalpy of +2.5 kcal/mote. The rate of helix dissociation of the A₆C₆U₆ hairpin is in the range of 10³ to lO⁵ sec^?1 with an activation enthalpy of 21 $\text{kcal}\text{mole}$. A comparison with the kinetic parameters obtained for A · U oligomer helices shows a specific influence of the C₆ loop due to the stacking tendency of the cytosine residues. This intluence is preferentially reflected in the relatively low value of the rate constant of helix formation.     

Stopped-flow Circular Dichroism: A rapid kinetic study of the binding of a sulphonamide drug to bovine carbonic anhydrase

The technique of Stopped-Flow Circular Dichroism allows the simultaneous monitoring of chiroptical and absorbance transients at millisecond time resolution. In the binding of a chromophoric sulphonamide to Bovine Carbonic Anhydrase, the rapid kinetics of the induced circular dichroism and difference spectra proceed in parallel with bimolecular rate constant k₁ = 5 × 10⁶ M^?1 sec^?1 and apparent half reaction time of 8.7 msec for 24 μM reactants. A single classical binding process is indicated by both optical parameters.     

Peroxidase-like activities of iron(III)—Porphyrins: kinetics of the reduction of a peroxidatically active derivative of deuteroferriheme by anilines

The kinetics of the reduction by aniline and a series of substituted anilines of a peroxidatically active intermediate, formed by oxidation of deuteroferriheme with hydrogen peroxide, have been studied by stopped-flow spectrophotometry. The reaction with aniline was first order with respect to [intermediate] and showed first-order saturation kinetics with respect to [aniline]. The second-order rate constant was 2.0 ± 0.2 × 10⁵ M^?1 sec^?1 at 25°C (independent of pH in the range 6.60–9.68) compared with the value of 2.4 × 10⁵ M^?1 sec^?1 for the reaction of aniline with horseradish peroxidase Compound I. The effect of aniline substituents upon reactivity towards the heme intermediate closely paralled those reported for reaction with the enzymic intermediate. Anilines bearing electron-donating substituents reacted more rapidly and those bearing electron-withdrawing substituents more slowly than the unsubstituted amine. The rate constants for the heme intermediate reactions (k_dfh)found to be related to those for the enzymic reactions (k_hrp) by the equation:log k_DFH= 0.65log k_HRP+ 1.96 with a correlation coefficient of 0. 98.