Simple Bimolecular Process Research Articles

Dependence of the Fe(II)-Gallic Acid Coordination Compound Formation Constant on the pH.

One important property of tannins involves their ability to form coordination compounds with metal ions, which is vital for the bioavailability of these ions, as well as for the antibacterial and antioxidative activities of tannins. In this study, the pH dependence of interactions between gallic acid, one of the basic building blocks of tannins, and Fe(II) ions, was investigated using UV/Vis spectroscopy, in conjunction with density functional theory (DFT) calculations. Moreover, two models were developed to explain the processes taking place in the solution. The first model treated the reaction as a simple bimolecular process while the second also considered the protolytic equilibrium, which was proven very successful in discerning the pH dependence of formation constants, and whose assumptions were well supported by DFT calculations. We showed that the two-time deprotonated gallic acid species forms the coordination compound with Fe(II) ions in a 1:1 molar ratio. To gain better insight into the process, the coordination compound formation was also studied using various DFT functionals, which further supported the model results. Furthermore, due to the relatively low sample amounts needed, the methodology developed here will be useful to study compounds that are more difficult to isolate.

Isoniazid Inhibits the Heme-Based Reactivity of Mycobacterium tuberculosis Truncated Hemoglobin N

Isoniazid represents a first-line anti-tuberculosis medication in prevention and treatment. This prodrug is activated by a mycobacterial catalase-peroxidase enzyme called KatG in Mycobacterium tuberculosis), thereby inhibiting the synthesis of mycolic acid, required for the mycobacterial cell wall. Moreover, isoniazid activation by KatG produces some radical species (e.g., nitrogen monoxide), that display anti-mycobacterial activity. Remarkably, the ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of (pseudo-)enzymatic detoxification systems, including truncated hemoglobins (trHbs). Here, we report that isoniazid binds reversibly to ferric and ferrous M. tuberculosis trHb type N (or group I; Mt-trHbN(III) and Mt-trHbN(II), respectively) with a simple bimolecular process, which perturbs the heme-based spectroscopic properties. Values of thermodynamic and kinetic parameters for isoniazid binding to Mt-trHbN(III) and Mt-trHbN(II) are K = (1.1±0.1)×10−4 M, k on = (5.3±0.6)×103 M−1 s−1 and k off = (4.6±0.5)×10−1 s−1; and D = (1.2±0.2)×10−3 M, d on = (1.3±0.4)×103 M−1 s−1, and d off = 1.5±0.4 s−1, respectively, at pH 7.0 and 20.0°C. Accordingly, isoniazid inhibits competitively azide binding to Mt-trHbN(III) and Mt-trHbN(III)-catalyzed peroxynitrite isomerization. Moreover, isoniazid inhibits Mt-trHbN(II) oxygenation and carbonylation. Although the structure of the Mt-trHbN-isoniazid complex is not available, here we show by docking simulation that isoniazid binding to the heme-Fe atom indeed may take place. These data suggest a direct role of isoniazid to impair fundamental functions of mycobacteria, e.g. scavenging of reactive nitrogen and oxygen species, and metabolism.

Influence of 2,5-dichloro-1,4-benzoquinone on jack bean urease activity. Inhibitory effect, total reducing capacity and DPPH radical scavenging activity.

Inhibition of jack bean activity by 2,5-dichloro-1,4-benzoquinone (DCBQ) was studied in phosphate buffer, pH 7.0. It was found that DCBQ acted as a strong, time and concentration dependent inactivator of urease. Under the experimental conditions obeyed the terms of pseudo-first-order reaction, urease was totally inactivated. Application of Wilson-Kitz method proved that the urease-DCBQ interaction followed a simple bimolecular process and the presence of intermediate complex was undetectable. The determined second order rate constant of the inactivation was 0.053 (μM min)(-1). Thiols such as l-cysteine, glutathione and dithiothreitol (DTT) protected urease from inhibition by DCBQ but DCBQ-modified urease did not regain its activity after DTT application. The thiol protective studies indicated an essential role of urease thiol(s) in the inhibition. The irreversibility of the inactivation showed that the process was a result of a direct modification of urease thiol(s) by DCBQ (DCBQ chlorine(s) substitution). The decomposition of DCBQ in aqueous solution at natural light exposure was monitored by visible spectrophotometry, determination of the total reducing capacity (Folin-Ciocalteu method) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging ability. The DCBQ conversion resulted in a decrease of the inhibition power and was well correlated with the increase of the total reducing capacity and DPPH scavenging ability. These findings were attributed to DCBQ transformation by photolysis and the hydrolysis effect was found to be negligible.

Binding and Electron Transfer between Cytochrome b5 and the Hemoglobin α- and β-Subunits through the Use of [Zn, Fe] Hybrids

We have measured the binding affinity (KA) and electron transfer (ET) rate constants (k) for the complex of hemoglobin (Hb) and cytochrome b5 (b5), using triplet quenching titrations of mixed-metal [ZnM, Fe3+(N3-)] Hb hybrids and of fully substituted Zn−mesoporphyrin (ZnM)Hb by b5 (trypsin-solubilized, bovine) (pH values 6.0 and 7.0). The use of the mixed-metal Hb hybrids with Zn in one chain type allows us to selectively monitor the 3ZnP → Fe3+P ET reaction of Fe3+b5 with either the α-chains or the β-chains. The self-consistent analysis of the results for the mixed-metal hybrids and those for the (ZnM)Hb allows us to determine the reactivity and affinity constants for the interactions of b5 with the individual subunits of T-state Hb. The results confirm that ET occurs within a complex between b5 and Hb, not through a simple bimolecular collision process. At pH 6.0, the binding affinity constant of the α-chains (Kα ≈ 2.0 × 104 M-1) is ∼4-fold larger than that of the β-chains (Kβ = 4.9 × 103 M-1); the intracomplex ET rate constant of the α-chains (kα ≈ 1500 s-1) is ∼2-fold larger than that of the β-chains (kβ ≈ 850 s-1). The binding affinity and ET rate constant of the α-chains both decrease as the pH is increased from 6.0 to 7.0; the binding affinity of the β-chains is essentially the same at pH 6.0 and 7.0, while the ET reactivity decreases. The kinetic results are consistent with a docking model in which each subunit binds a molecule of b5. However, they permit an alternative in which b5 reacts with the α-chains by binding at a site which spans the α1β2 dimer interface.

Comparison of the reactivity of (CO)3L2ReOR, (CO)3L2ReOAr and (CO)3L2ReNHAr with carbon dioxide and other electrophiles

The reactivity of a series of low-valent Re alkoxides, aryloxides, and arylamides with CO[sub 2], CS[sub 2], and other electrophiles has been investigated. The methoxide complexes fac-(CO)[sub 3](L[sub 2])ReOCH[sub 3] (2a, L = PMe[sub 3]; 2b, L[sub 2] = 1,2-(AsMe[sub 2])[sub 2]C[sub 6]H[sub 4] (diars)) react with activated organic carbonyl compounds such as acid chlorides, anhydrides, and activated esters to give products resulting from cleavage of the Re-OCH[sub 3] bond. The methoxide complexes react rapidly with CO[sub 2] and CS[sub 2] to give the expected insertion products fac-(CO)[sub 3](L[sub 2])ReX(CX)OCH[sub 3] (8, X = O; 9, X = S). The analogous aryloxides 1 do not react with CO[sub 2] and only the PMe[sub 3]-substituted system reacts with CS[sub 2]. In the latter transformation fac,cis-(CO)[sub 3](PMe[sub 3])[sub 2]ReOC[sub 6]H[sub 4]CH[sub 3] leads to the isomerized product mer,trans-(CO)[sub 3](PMe[sub 3])[sub 2]ReS(CS)OC[sub 6]H[sub 4]CH[sub 3]. The arylamide fac-(CO)[sub 3](depe)ReNHC[sub 6]H[sub 5] (3c) reacts with CO[sub 2] to give the metallaurethane 10C. The metallacarbonates 8 were found to be quite reactive. They readily deinsert CO[sub 2] to reform the methoxide complexes. Hydrolysis of 8b leads to a bridging carbonate complex (CO)[sub 3](diars)Re-([mu]-CO[sub 3])-Re(diars)(CO)[sub 3], whose structure has been determined by an X-ray diffraction study.more » Kinetic studies on the formation of 9b suggest that the reaction of 2b with CX[sub 2] is a simple bimolecular process. 41 refs., 3 figs., 4 tabs.« less

A generalized theoretical treatment of the kinetics of an enzyme-catalysed reaction in the presence of an unstable irreversible modifier

A generalized theoretical treatment of the kinetics of an enzyme-catalysed reaction in the presence of an unstable irreversible inhibitor (or activator) is presented. Analytical expressions describing the time-dependence of product formation have been derived in coefficient form amenable to non-linear regression analysis for two operationally distinct types of reaction mechanism dependent on whether the reaction of the unstable modifier (X) with either or both the free enzyme (E) and enzyme-substrate complex (ES) occurs as a simple bimolecular process, or proceeds through the intermediacy of either or both adsorptive enzyme-modifier (EX) and enzyme-modifier-substrate (EXS) complexes in what may be considered as an extension of the Botts-Morales general modifier mechanism for (stable) reversible enzyme inhibitors and activators. Special cases of both models are classified in an analogous way to the traditional naming of reversible enzyme modifications, and guidelines concerning tests of mechanism and determination of kinetic parameters are given. In particular, it has been shown that kinetic constants describing enzyme inactivation by an unstable site-specific inhibitor forming a reversible EX complex prior to covalent modification step may be determined from a single progress curve. Kinetic analysis of the extended Botts-Morales mechanism describing irreversible enzyme inactivation has demonstrated that analytical expressions describing the time-course of product formation may be derived for a stable modifier by retaining the usual steady-state assumptions regarding the fluxes around ES and EXS provided quasi-equilibrium modifier binding to E and ES is assumed, but for unstable modifiers all of the binding steps must be assumed to be at quasi-equilibrium in the steady-state, except under restrictive circumstances.

Transient Kinetics of Heparin-catalyzed Protease Inactivation by Antithrombin III: Characterization of assembly, product formation, and heparin dissociation steps in the factor Xa reaction

The kinetics of alpha-factor Xa inhibition by antithrombin III (AT) were studied in the absence and presence of heparin (H) with high affinity for antithrombin by stopped-flow fluorometry at I 0.3, pH 7.4 and 25 degrees C, using the fluorescence probe p-aminobenzamidine (P) and intrinsic protein fluorescence to monitor the reactions. Active site binding of p-aminobenzamidine to factor Xa was characterized by a 200-fold enhancement and 4-nm blue shift of the probe fluorescence emission spectrum (lambda max 372 nm), 29-nm red shift of the excitation spectrum (lambda max 322 nm), and dissociation constant (KD) of about 80 microM. Under pseudo-first order conditions [( AT]0, [H]0, [P]0 much greater than [Xa]0), the observed factor Xa inactivation rate constant (kobs) measured by p-aminobenzamidine displacement or residual enzymatic activity increased linearly with the antithrombin concentration (i.e. corrected for probe competition) up to 300 microM in the absence of heparin, indicating a simple bimolecular process with a rate constant of 2.1 x 10(3) M-1 s-1. In the presence of heparin, a similar linear dependence of kobs on effective AT.H complex concentration was found up to 25 microM whether the reaction was followed by probe displacement or the quenching of AT.H complex protein fluorescence due to heparin dissociation, consistent with a bimolecular reaction between AT.H complex and free factor Xa with a 300-fold enhanced rate constant of 7 x 10(5) M-1 s-1. Above 25 microM AT.H complex, an increasing dead time displacement of p-aminobenzamidine and a downward deviation of kobs from the initial linear dependence on AT.H complex concentration were found, reflecting the saturation of an intermediate Xa.AT.H complex with a KD of 200 microM and a limiting rate of Xa-AT product complex formation of 140 s-1. Kinetic studies at catalytic heparin concentrations yielded a kcat/Km for factor Xa at saturating antithrombin of 7 x 10(5) M-1 s-1 in agreement with the bimolecular rate constant obtained in single heparin turnover experiments. These results demonstrate that 1) the accelerating effect of heparin on the AT/Xa reaction is at least partly due to heparin promoting the ordered assembly of antithrombin and factor Xa in an intermediate ternary complex and that 2) heparin catalytic turnover is limited by the rate of conversion of the ternary complex intermediate to the product Xa-AT complex with heparin dissociation occurring either concomitant with this step or in a subsequent faster step.

The reduction of lumiflavin by EDTA radicals and evidence for association of EDTA with flavinsemiquinone

Lumiflavin was reduced by the solvated electron, .CO - 2 radicals and EDTA radicals in the presence of 0.01–0.05 M EDTA. The three reductants produced Fl intermediates with the same absorption spectrum and which decayed by a first order process with a t 1 2 of ∼400 μs . This was in contrast to .F1H intermediates in the absence of EDTA, which exhibited a different absorption spectrum and decayed by second order kinetics. These changes were attributed to complexation with EDTA of the .F1H semiquinone formed by the reducing radicals. Complexation did not alter the stoichiometric yield of F1H - from the semiquinone decay, a feature which agreed with previous photochemical studies. The reduction by EDTA radicals was not a simple bimolecular process and occurred on a much longer (∼150μs) time scale than for the other radicals. The complex kinetics of the growth was in line with predictions based on the mechanism of reaction of EDTA radicals reported by Armstrong, Hemmerich and Traber ( Photochem. Photobiol. 1982, 35, 747), but there are further complicating factors which need to be considered.

Characterization of tachykinin receptors in isolated basilar arteries of guinea‐pig

In circular lengths cut from the basilar artery of guinea-pig (0.2-0.3 mm o.d.) relaxations induced by substance P and neurokinin A were highly susceptible to mechanical damage of the endothelium by rubbing. The precontraction induced by prostaglandin F2 alpha but not that of 124 mM potassium was reduced considerably by the rubbing procedure. Concentration-dependent relaxations were evoked by tachykinin agonists in the following order of potency: substance P = physalaemin greater than neurokinin A greater than eledoisin. Physalaemin was, however, a partial agonist, giving only half the maximum relaxation as compared to the other tachykinins. The two putative tachykinin receptor antagonists, spantide ([D-Arg1, D-Trp7,9, D-Leu11] substance P) and [D-Pro2, D-Trp7,9] substance P, shifted the concentration-dependent relaxations of substance P to the right in a parallel manner. Calculation of pA2 values and Schild plot analysis revealed pA2 values of 7.4-7.6 for spantide and 6.9-7.0 for [D-Pro2, D-Trp7,9] substance P, irrespective of whether substance P or neurokinin A was used as agonist. The pA2 values and the Schild plot analysis suggest a specific interaction between tachykinin agonists and antagonists that follow a simple bimolecular process. The results suggest the presence of tachykinin receptors of the 'SP-P' type in guinea-pig basilar arteries which, for induction of relaxation, involves the release of an endothelium-derived relaxing factor.

Beta nerve growth factor binding to PC12 cells. Association kinetics and cooperative interactions.

The association kinetics of 125I beta nerve growth factor (NGF) binding to the PC12 clonal cell line have been examined in detail at 0.5 and 37 degrees C. These data were examined by utilizing a reversible second-order integrated rate equation, and the results were not consistent with a simple bimolecular process. Two association rates were required to explain the results adequately. At 37 degrees C, the faster component was estimated to have a second-order association rate constant of 1.4 X 10(7) M-1 s-1, while the rate constant for the slower component (3.8 X 10(6) M-1 s-1) was about 4-fold lower. As shown by others, the temperature dependence of the dissociation kinetics indicated that while the rapidly dissociating component was only slightly slowed by lowering the chase temperature to 0.5 degrees C, the second component was slowed by about 270-fold, from 8 X 10(-4) s-1 to 3 X 10(-6) s-1. The binding data that describe the slowly dissociating component were obtained by utilizing this differential temperature dependence and revealed a concave downward Scatchard plot. The binding parameters determined from computer analysis using a nonlinear fitting program (LIGAND) suggest that this component consists of (a) an interacting class of about 4000 sites/cell that have a first stoichiometric steady-state dissociation constant of 65 pM and a second stoichiometric interaction constant of 16 pM, indicative of positively cooperative interactions, and (b) a class of sites consistent with a ratio of sites/Kd of about 11.1 sites/(cell X pM). The steady-state binding results at 37 degrees C indicated only one class of binding sites (155,000 +/- 18,000 sites/cell) that had an apparent Kd of 0.52 +/- 0.03 nM. One class of sites was also observed at 0.5 degrees C, and the receptor concentration was found to be reduced (99,000 +/- 7600 sites/cell) while the Kd was increased (1.7 +/- 0.14 nM). A significant level of positively cooperative interactions was observed frequently at 37 degrees C that was not due to a failure to reach steady-state conditions, internalization, or degradation. Since cooperativity of binding was never observed at 0.5 degrees C, a membrane event may be involved. Determination of the contribution of the different classes of NGF receptors found on PC12 cells to the biological actions of NGF requires a clear understanding of their kinetic properties and their relationship to each other. The studies presented here indicate that their interactions are more complex than previously described.

Kinetics of carbon monoxide binding to phenobarbital-induced cytochrome P-450 from rat liver microsomes: a simple bimolecular process.

The kinetics of carbon monoxide binding to phenobarbital-induced cytochrome P-450 (P-450PB) and to its enzymatically inactive form P-420PB have been investigated by both stopped-flow and flash-photolysis spectrophotometry. When the simultaneous presence of both forms of the enzyme is taken into account, the binding of CO to these two proteins can be described in terms of two bimolecular processes with rate constants of 4.5 X 10(6) M-1.S-1 and 4.7 X 10(5) M-1.S-1 for P-450PB and 1.7 X 10(7) M-1.S-1 and 1.5 X 10(6) M-1.S-1 for P-420PB. From kinetic studies of the binding of CO to P-450PB under different experimental conditions, investigations of the homogeneity of our P-450PB preparations, and comparative kinetic investigations of P-450s from different sources, we conclude that CO binding to reduced P-450PB is a simple bimolecular process and that the observed biphasic traces are due to heterogeneity of the proteins. This conclusion is in contrast with previous reports of complex reaction mechanisms for the binding of CO to P-450PB. Optical spectroscopy studies indicate the existence of an equilibrium between P-450PB and P-420PB, at least for the reduced carbonyl derivatives of the enzymes. The interconversion is strongly influenced by the aggregation state of the protein. Large differences between the CO binding properties of P-450PB and those of P-420PB are found. These are discussed in terms of possible effects of the proximal ligation state of the iron on heme reactivity.

Mechanism of acetoacetylation of substituted anilines

Kinetics have been studied of the reaction of diketene with substituted anilines, and the reaction rate has been found to depend on relative permittivity of the system. The rate and equilibrium constants have been calculated by combination of rate and equilibrium relations with the relation by Amis; the constants correlate with the Hammett substituent constants. The reaction does not proceed as a simple bimolecular process. A reaction mechanism has been suggested.

Irreversible inhibition of phosphotransacetylase by S-dimethylarsino-CoA

S-Dimethylarsino-CoA was synthesized by acylation of CoA with dimethylchloroarsine. The new analogue of acetyl-CoA was tested as an active-site-directed irreversible inhibitor of phosphotrasacetylase (EC. 2.3.1.8), carnitine acetyltransferase (EC 2.3.1.7) and citrate synthase (EC 4.1.3.7). Irreversible inhibition was observed only with phosphotransacetylase, which was derivatized via a simple bimolecular process ( k 2 = 197 ± 15 min −1· M −1 ). Acetyl-CoA provided complete substrate protection against the inactivation, while phosphate (a substrate) and desulfo-CoA (a competitive inhibitor) provided a partial protection. The inactivation was not reversed by dithiothreitol. The new reagent was a linear competitive inhibitor versus acetyl-CoA with both carnitine acetyltransferase ( K i = 41 μM) and citrate synthase ( K i = 20 μM). Chemical studies showed that S-dimethylarsino-CoA reacts with the thiol of N α -acetylcysteine but not with the side-chain functional groups of histidine and lysine. The nature of the chemical modification of cysteine was determined by investigating a model system. Thus the chemical reaction between the thioarsenite linkage of S-dimethylarsinobenzylmercaptan and the thiol of cysteine was shown to involve transesterification of the dimethylarsino group.

Dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis: The oxidation reaction

The oxidation by ferricyanide of the dimeric (HbI) and tetrameric (HbII) hemoglobins from the bivalve mollusc Scapharca inaequivalvis has been studied in static and kinetic experiments. Both hemoglobins give rise to hemichromes as stable oxidation products. Oxidation of deoxyHbI yields a hemichrome by a simple bimolecular process. No intermediate Met form can be detected during the reaction even in rapid mixing experiments. The HbI hemichrome undergoes a reversible pH-dependent dissociation into monomers. A simple model has been proposed to account for the linkage between proton binding and subunit dissociation. In the case of tetrameric HbII, oxidation yields an intermediate Met form. Thus, the kinetics of the oxidation reaction are always biphasic; the fast reaction is a bimolecular process and yields the Met derivative. The slow reaction is a monomolecular process and corresponds to the conversion of the Met form into the hemichrome: its rate is independent of the state of ligation of the ferrous protein and decreases with increase of pH. The HbII hemichrome is tetrameric when newly formed: it tends to dissociate into lower molecular weight species with the same optical properties. The rate of dissociation is relatively fast at neutral pH ( t 1 2 ≈ 12 min) and markedly less at alkaline pH values. The HbI and HbII hemichromes are reduced by dithionite yielding the spectra of the native deoxygenated proteins: in the case of HbII, the tetrameric structure of the native protein is re-acquired.

Equilibrium and kinetic studies of oxygen binding to the haemocyanin from the freshwater snail Lymnaea stagnalis.

The binding of oxygen by the haemocyanin of the gastropod Lymnaea stagnalis was studied by equilibrium and kinetic methods. The studies were performed under conditions in which the haemocyanin molecule was in the native state. Over the pH range 6.8-7.6, in the presence of 10mM-CaCl2 the haemocyanin bound O2 cooperatively. Over this pH range the haemocyanin molecule displayed a normal Bohr effect whereby the O2 affinity of the molecule decreased with a fall in the pH of the solution. The maximum slope of the Hill plot (hmax.) was 3.5, obtained at pH 7.5. An increase in the CaCl2 concentration from 5 to 20 mM at pH 6.8 resulted in a slight increase in the oxygen affinity, with hmax. remaining virtually unchanged. At constant pH and CaCl2 concentration, an increase in NaCl concentration from 0 to 50 mM resulted in a small decrease in O2 affinity, but a significant increase in the value of hmax. from 3.5 to 8.6. Temperature-jump relaxation experiments over a range of O2 concentrations produced single relaxation times. The dependence of the relaxation time on the reactant concentrations indicated a simple bimolecular binding process. The calculated association and dissociation rate constants for this process at pH 7.5 are 29.5 X 10(6) M-1 X S-1 and 49 S-1 respectively. The association rate constant kon was found to be essentially independent of pH and CaCl2 concentration. The dissociation rate constant, koff, however, increased with a decrease in the pH, but was also independent of CaCl2 concentration. These results indicate that the stability of the haemocyanin-O2 complex is determined by the dissociation rate constant.

The role of cysteine residues in the catalytic activity of glycerol-3-phosphate dehydrogenase

Glycerol-3-phosphatedehydrogenase ( sn- glycerol-3- phosphate: NAD + 2-oxidoreductase, EC 1.1.1.8) has been shown to be sensitive to inhibition by iodoacetate. The reaction of the enzyme with iodoacetate, which appears to be a simple bimolecular process, is accompanied by a corresponding loss of enzyme activity. In addition to changes in activity, the alkylation reaction was monitored by the incorporation of radioactivity from iodo[2- 14C]acetate, by changes in amino acid composition, and by changes in the content of free sulfhydryl groups. It is concluded that there are two cysteine residues in the native dimeric enzyme which are essential for enzymic activity. The rate of inactivation was relatively insensitive to the presence of various compounds with the exception of NADH which markedly inhibited the reaction. Kinetic and binding studies showed that the binding of NADH prevents alkylation and, conversely, alkylation prevents NADH binding. From the pH dependence of the alkylation reaction, the pK a of the essential sulfhydryl groups was calculated to be 8.5 and it is suggested that the binding of coenzyme is independent of the state of ionization of these groups.

Mechanistic studies of glutamine synthetase from Escherichia coli: kinetics of ADP and orthophosphate binding to the unadenylylated enzyme.

The kinetics of protein fluorescence change exhibited by ADP or orthophosphate addition to the Mg2+-or Mn2+-activated unadenylylated glutamine synthetase from Escherichia coli were studied. The kinetic patterns of these reactions are incompatible with a simple bimolecular binding process and a mechanism which required protein isomerization prior to substrate binding. They are consistent with a mechanism in which direct substrate binding is followed by a substrate-induced conformational change step, ES in equilibrium ES. At pH 7.0 and 15 degrees C, the association constants for the direct binding (K1) of ADP to MnE1.0 and of Pi to MnE1.0ADP are 3.9 X 10(4) and 2.28 X 10(2) M(-1), respectively. The association constant for the direct binding of ADP to MnE1.0Pi is 2.3 X 10(4) M(-1) at pH 7.0 and 19 degrees C. The deltaG degrees for the substrate-induced conformational step are -3.5 and -1.3 kcal mol(-1) due to ADP binding to MnE1.0Pi and MnE1.0, respectively, and -1.4 kcal mol(-1) due to Pi binding to MnE1.0ADP. Rate constants, k2 and k(-2), for the isomerization step are: 90 and 9.5 s(-1) for ADP binding to MnE1.0, 440 and 0.36 s(-1) for ADP binding to MnE1.0Pi, and 216 and 1.8 s(-1) for Pi binding to MnE1.0ADP. Due to low substrate affinity, the association constant for direct Pi binding to MnE1.0 was roughly estimated to be 230 M(-1) and k2 = 750 s(-1), k(-2) = 250 s(-1). At 9 degrees C and pH 7.0, the estimated association constants for the direct ADP binding to MgE1.0 and MgE1.0 Pi are 1.8 X 10(4) and 1.6 X 10(4) M(-1), respectively; and the rate constants for the isomerization step associated with the corresponding reaction are k2 = 550 s(-1), k(-2) = 500 s(-1), and k2 = 210 s(-1), k(-2) = 100 s(-1). From the kinetic analysis it is evident that the inability of Mn2+ to support biosynthetic activity of the unadenylylated enzyme is due to the slow rate of ADP release from the MnE1.0PiADP complex. In contrast the large k(-2) obtained for ADP release from the MgE1.0ADP or MgE1.0PiADP complex indicates that this step is not rate limiting in the biosynthesis of glutamine since the k catalysis obtained under the same conditions is 7.2 s(-1).

The magnesium ion-dependent adenosine triphosphatase of myosin. Two-step processes of adenosine triphosphate association and adenosine diphosphate dissociation.

The kinetics of protein-fluorescence change when rabbit skeletal myosin subfragment 1 is mixed with ATP or adenosine 5'-(3-thiotriphosphate) in the presence of Mg(2+) are incompatible with a simple bimolecular association process. A substrate-induced conformation change with DeltaG(0)<-24kJ.mol(-1) (i.e. DeltaG(0) could be more negative) at pH8 and 21 degrees C is proposed as the additional step in the binding of ATP. The postulated binding mechanism is M+ATPright harpoon over left harpoonM.ATPright harpoon over left harpoonM*.ATP, where the association constant for the first step, K(1), is 4.5x10(3)m(-1) at I 0.14m and the rate of isomerization is 400s(-1). In the presence of Mg(2+), ADP binds in a similar fashion to ATP, the rate of the conformation change also being 400s(-1), but with DeltaG(0) for that process being -14kJ.mol(-1). The effect of increasing ionic strength is to decrease K(1), the kinetics of the conformation change being essentially unaltered. Alternative schemes involving a two-step binding process for ATP to subfragment 1 are possible. These are not excluded by the experimental results, although they are perhaps less likely because they imply uncharacteristically slow bimolecular association rate constants.

Properties of Modified Cytochromes: II. LIGAND BINDING TO REDUCED CARBOXYMETHYL CYTOCHROME c

SUMMARY Reduced (Fe”+) carboxymethylated cytochrome c (Cmcytochrome c) reversibly binds ligands of ferrous iron, e.g. CO, cyanide, ethyl isocyanide, and oxygen, at neutral PH. Titrations of the reduced protein with CO show that over the pH range 6 to 9.5 the stoichiometry of binding is one, and that the spectrum of the Cm-cytochrome c complex with CO is practically pH-independent. The kinetics of CO binding of Cm-cytochrome c have been studied as a function of pH. At low pH values the binding process as observed by stopped flow or photolysis techniques conforms to a simple bimolecular process (2’ = 1.6 X 106M-r s-l), whereas at high pH the process, although remaining simple, it is NlOO-fold slower. At intermediate pH values, the binding of CO occurs in a biphasic reaction, the proportions of the fast and slow phases depending on the pH, on the monitoring wave length, and, in the case of photolytic experiments, on the CO concentration. The dissociation rate constants of CO from Cm-cytochrome c were measured by replacement with oxygen or ethyl isocyanide. A model is proposed in which the acid and alkaline conformers of the ligand-free protein have different ligandbinding properties. It is suggested that there is a pH-dependent transition in the CO adduct of Cm-cytochrome c and the pK of this transition is reported. The model is consistent with simple thermodynamic considerations. Binding between ferrous Cm-cytochrome c and 02 has been examined. At pH 6, a stable O2 complex, similar to myoglobin, is formed, whereas at pH 9 0% rapidly oxidized the heme iron.

Kinetics and mechanisms of cleavage of allylic derivatives of group IVA elements by mercuric salts I. Allylsilanes

The kinetics and mechanism of cleavage of trimethylallyl silane and its derivatives by mercuric salts have been investigated in solvent acetonitrile. The reaction was followed by spectrophotometric and gas chromatographic methods. The stoichiometry of the reaction is not that of a simple bimolecular process, and is best represented by the reaction scheme: ▪ A second molecule of substrate is rapidly attacked by Me 3SiX formed in the rate determining step. Separate examination of (2) revealed that reaction occurred in the absence of mercuric salts. Me 3SiX was not consumed during the process but acts as a catalyst, resulting in first order kinetic behaviour. Mercuric salts strongly catalyse this step, acting as Lewis acids, and enhancing the already polar nature of Me 3SiX. Rate constants were calculated for (1) from chromatographic data, adopting the above reaction scheme. The reaction was found to be first order in both substrate and electrophile. Spectrophotometric studies indicated that (1) proceeded via a π complex, the rate of decomposition of which corresponded with that obtained by chromatographic techniques, thus supporting the proposed mechanism. In addition, evidence is presented which supports an S E2 rather than S F transition state for (1).