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PubMed1977

[Influence of pH on the steady state kinetics of electron transfer through the cytochrome chain of submitochondrial particles. Kinetic model for regulating the activity of carriers by the local concentration of hydrogen ions in the membrane].

The kinetic parameters of the submitochondrial particles cytochrome chain obtained from steady-state kinetics were studied for pH dependence. The life-times of the activated states (tau) for cytochrome pairs b leads to c1 and a leads to a3 are shown to bear dissimilar dependence on pH of the medium, while for cytochrome pairs c1 leads to c and c leads to a they display practically no pH dependence at all. The rate constants of the non-activated state (alphai-kiCo) decreased for the pair b leads to c1 and increased for a leads to a3 with the increase of pH from 6.5 to 8.5. The apparent pK values obtained therefrom were 7.2 and 8.9, respectively. A kinetic model is proposed suggesting that local pH in the mitochondrial membrane, dependent on the rate of electron transfer, may be a controlling factor for the ratio of activated and non-activated carrier states. The model is in good consistence with the experimental dependences of k'i on V and the pH dependences of alpha2 for b leads to c1 and a leads to a3. It also gives a qualitative prediction for the pH dependences of the ordinate intercepts of the straight lines in l/(k'i--alphai) vs. l/V plots. The rate constants for the diffusion of hydrogen and hydroxyl ions in the membrane are estimated on the basis of our kinetic data to be 10(4)--10(5) s-1 and 10(2)--10(3) s-i, respectively.

Cytochromes↗

PubMedDate not supplied

Apparent aberrancy in the kinetics of intracellular metabolism of a single substrate by two enzymes. An alternative explanation for anomalies in the kinetics of sulfation and glucuronidation.

The intracellular metabolism of a substrate by two simultaneous, competing metabolic routes in intact isolated cells is analyzed by use of a kinetic model. The following assumptions were made: 1) uptake and back-transport to the medium of the unchanged substrate are symmetrical and linear with the extracellular and intracellular concentrations, respectively; 2) two metabolic routes convert the substrate simultaneously, according to Michaelis-Menten kinetics, from the same intracellular substrate pool. Equations describing transport and metabolism of the substrate are derived for a steady-state situation. It is shown that the intracellular substrate concentration increases more than proportionally with the extracellular concentration when the metabolic reaction with high affinity becomes saturated. This causes the conversion via unsaturated routes (low-affinity conversion) also to increase more than proportionally. Thus, the kinetics of metabolism show anomalous behavior when related to the extracellular substrate concentration. Furthermore, the model suggests that the apparent KM value of a metabolic conversion in an intact cell, expressed and determined as the extracellular substrate concentration at half the maximal velocity of the reaction, is not a true Michaelis-Menten constant; its value is determined by the kinetic parameters of competing reactions. Some results from the literature on anomalous behavior of sulfation and glucuronidation in different preparations have been analyzed with our model; a good agreement was obtained between experimental results and values determined by extrapolation according to our model.

Biological Transport↗

PubMed1982

The kinetic mechanism of D-amino acid oxidase with D-alpha-aminobutyrate as substrate. Effect of enzyme concentration on the kinetics.

The kinetic mechanism of hog kidney D-amino acid oxidase with D-alpha-aminobutyrate as substrate has been examined in detail using a combination of steady state and rapid reaction methods. At concentrations of D-alpha-aminobutyrate below 0.5 mM, the rapid reaction and steady state results are consistent with the mechanism previously proposed for D-alanine (Massey, V., and Gibson, Q. H. (1964) Fed. Proc. 23, 18-29; Porter, D. J. T., Voet, J. G., and Bright, H. J. (1977) J. Biol. Chem. 252, 4464-4473). Both flavin reduction by D-alpha-aminobutyrate and reoxidation are quite rapid. Release of product from the oxidized enzyme has been measured directly and matches the turnover number at infinite concentrations of both substrates. Substitution of deuterium for the alpha-hydrogen decreases the rate of reduction 1.4-fold, without any effect on the apparent Kd. Computer simulations show that the kinetic isotope effects on the reductive half-reaction with D-alanine reported by Porter et al. (see above reference) can be explained using a two-step model with a kinetic isotope effect of 1.75 on the limiting rate of reduction. The effect of enzyme concentration on the kinetics has been examined in some detail. With D-alanine as substrate, increasing the enzyme concentration over the range 29 nM to 17 microM resulted in less than a 2-fold decrease in the turnover number. The Kd for benzoate binding also decreased marginally with increasing enzyme concentration. The effect of enzyme concentration is consistent with a decrease in the rate of release of ligands from the oxidized enzyme as the enzyme concentration is increased.

Aminobutyrates↗

PubMedDate not supplied

[Kinetics of poly-enzyme system reactions. II. Nonsteady-state kinetics. Presteady-state and relation modes in a bi-enzyme system and linear sequences].

Kinetic aspects of reactions in homogeneous multienzyme systems under nonsteady state conditions were investigated. An analysis of formal-kinetic relationships, describing the time course of system was conducted with a bienzyme system. Presteady state kinetics of processes in lineal multienzyme systems was investigated. Relax-kinetics methods were applied for the analysis of processes in lineal sequences. Methods of determination of number of stages initial substrate transformations and of number of enzymes were developed as well as methods for the analysis of sequences of intermediates in reaction pathway. Methods of determination of Vmax and Kmax for each individual enzyme are considered.

Enzymes↗

PubMed1993

Kinetic and regulatory mechanisms for (Escherichia coli) homoserine dehydrogenase-I. Equilibrium isotope exchange kinetics.

Isotope exchange kinetics at chemical equilibrium were used to probe the mechanisms of substrate binding and regulatory behavior of homoserine dehydrogenase-I from Escherichia coli. At pH 9.0, 37 degrees C, Keq = 100 (+/- 20) for the catalyzed reaction: L-aspartate-beta-semialdehyde + NADPH + H+ = L-homoserine + NADP+. Saturation curves for the exchange reactions, [14C]L-homoserine <--> L-aspartate-beta-semialdehyde and [3H]NADP+ <--> NADPH were observed as a function of different reactant-product pairs, varied in constant ratio at equilibrium. The NADP+ <--> NADPH exchange rate was inhibited upon variation of pairs involving L-aspartate-beta-semialdehyde and L-homoserine, consistent with preferred order random binding of cofactors before amino acids. Optimal rate constants, derived by simulations of equilibrium isotope exchange kinetics data with the ISOBI program, indicate faster dissociation of amino acids than cofactors from the central complexes but nearly equal rates for association of cofactors and amino acids to free enzyme. Rate limitation of net turnover in both directions is determined by dissociation of cofactor from the E-cofactor complex. The allosteric modifier, L-threonine, produces distinctive perturbations of the saturation curves for isotope exchange, which were analyzed systematically with the ISOBI program. The best fit to the data was obtained by L-threonine inhibiting catalysis between the central complexes without altering substrate association-dissociation rates. Simulations also showed that rate-limiting catalysis suppresses the kinetic inhibition effects that are characteristic of preferred order substrate binding, producing patterns typical for a (rapid equilibrium) random kinetic scheme.

Catalysis↗

PubMed1996

A gas-liquid system for enzyme kinetic studies of volatile organic chemicals. Determination of enzyme kinetic constants and partition coefficients of trichloroethylene.

A gas-liquid system was developed for enzyme kinetic study with volatile organic chemicals (VOCs) by modification of the gas uptake method for the in vivo physiologically based pharmacokinetic experiment. This gas-liquid system, designed in our laboratory, is composed of: 1) a diffusion chamber for adjusting initial vapor concentration by mixing ambient air and the VOCs; 2) a condenser for maintaining the liquid level in the incubation chamber; 3) a stainless-steel metal bellows pump for recirculating vapor in this system; 4) a gas chromatograph equipped with an autosampler and a flame ionization detector; and 5) a computer for controlling automation and data processing. Trichloroethylene (TCE) was used as a model chemical, and enzyme kinetics were studied by measuring the depletion of TCE in the gas phase of the system. TCE-at initial concentrations of 56, 620, and 1240 ppm-was incubated with rat liver microsomes and a NADPH regenerating system in a 100-ml round-bottom flask. Based on parallel enzyme assays using p-nitrophenol as a substrate, cytochrome P450IIE1, activity remained stable up to 3 hr under the incubation conditions (37 degrees C and pH 7.4) whereas addition of glutathione into the incubation mixture did not affect TCE metabolism. Kinetic constants were analyzed using a two-compartment pharmacokinetic model and the computer software SimuSolv. Statistical optimization using the maximum-likelihood method produced apparent in vitro Vmax and KM values of 0.55 nmol/mg protein/min and 0.9 microM, respectively. In addition, this newly developed methodology has a number of advantages over those reported in the literature, including the potential utility of determining tissue partition coefficients of VOCs for physiologically based pharmacokinetic modeling. We conclude that this gas-liquid system is suitable for determination of kinetic constants near realistic environmental concentrations of VOCs including TCE.

Animals↗

PubMed1995

Kinetic evaluation of nonlinear drug elimination by a disposition decomposition analysis. Application to the analysis of the nonlinear elimination kinetics of erythropoietin in adult humans.

The disposition-decomposition analysis (DDA) methodology enables isolation of the overall elimination and distribution effects in pharmacokinetics and facilitates analysis which focuses on drug elimination kinetics and does not require a specific structured modeling of drug distribution processes. A computer algorithm enables a curve fitting and a kinetic estimation by integration of the convolution type integrodifferential equation in the DDA. The approach is demonstrated in an analysis of the nonlinear disposition kinetics of erythropoietin (Epo) in 10 healthy, adult human subjects who each received 10, 100, and 500 U/kg i.v. bolus doses of Epo. The nonlinearity is analyzed according to a Michaelis-Menten type nonlinear elimination function, considering simultaneous fitting to the data from all three doses in each subject. The simultaneous fittings produced estimates of the Michaelis-Menten parameters (mean, % cv) Vm (901 mU/mL/h, 19.4%) and km (4814 mU/mL, 24.6%). A linear clearance parameter is defined as the asymptotic clearance value approached when the drug level decreases toward zero. The degree of nonlinearity reached from various dosings was quantified in terms of a clearance ratio which is defined as the ratio between the linear clearance and the clearance estimated for the maximum drug concentration encountered at the given dose level. The subjects showed very little nonlinearity at the 10 U/kg dosing with a mean clearance ratio of 1.07 (2.1% CV) A statistically significant increase in the degree of nonlinearity was observed in the Epo elimination kinetics as the dosing level was increased to 100 and 500 U/kg, reaching clearance ratios of 1.66 (14% CV) and 4.33 (27% CV), respectively. A zero value for the global elimination rate parameter in all 30 dosings indicates that Epo's elimination is entirely accounted for by nonlinear pathway(s).

Adult↗

PubMed1996

Rat to human extrapolation of HCFC-123 kinetics deduced from halothane kinetics: a corollary approach to physiologically based pharmacokinetic modeling.

The goal of this study was to develop a human physiologically based pharmacokinetic (PBPK) model for the chemical HCFC-123 (2,2-dichloro-1,1,1-trifluoroethane) and its major metabolite, trifluoroacetic acid (TFA). No human kinetic data for HCFC-123 are available, thus a corollary approach was developed. HCFC-123 is a structural analog of the common anesthetic agent halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) and follows a common pathway of oxidative biotransformation, resulting in the formation of the same metabolite, TFA. In this study, halothane models for rats and humans were developed and validated. Then the corollary approach was used to develop a human HCFC-123 model from a rat HCFC-123 model. This strategy was implemented by using a previously validated PBPK model for HCFC-123/TFA in the Fisher 344 rat as a template model for halothane in rats. Model predictions were then compared to, and were in good agreement with, measured values for the concentration of halothane in rat blood and fat tissue. A human PBPK model for halothane was developed. The identical mode structure (with the exception of the description for the fat compartment) that was used to describe halothane and TFA in the rat was used for describing halothane and TFA in the human. Human physiological parameters for tissue volumes and flows were taken from the literature, and human tissue partition coefficients for halothane were measured in the laboratory. Based on reported similarity in metabolism of halothane by humans and rats, metabolic constants for halothane in the rat were used in the human model, and specific parameters describing the kinetics of TFA were estimated by optimization. The model was validated against human exposure data for halothane from six published studies (expired breath concentrations of halothane and serum/urine data for TFA). A similar approach was then used to derive a human HCFC-123 model for humans from the HCFC-123 rat model. The corollary approach described here illustrates the innovative use of template model structures to aid in the development and validation of models for structural analogs with similar metabolism and activity in biologic systems. Furthermore, given that the PBPK model adequately describes the kinetics of halothane in rats and humans and of HCFC-123 in rats, use of the human PBPK model is proposed for deriving dose-response estimates of human health risks in the absence of human kinetic data.

Animals↗

PubMed1990

Comparison of non-kinetic and kinetic approaches to individualization of gentamicin dosage.

A prospective study was carried out in 40 acutely ill patients to compare the non-kinetic and kinetic approaches to individualization of the dosage regimen of gentamicin. The patients were divided into two equal groups. For the non-kinetic group, the doses were derived from the physician's personal experience, on a mg/kg basis, and by use of nomograms. The total daily dose ranged from 1.43 to 4.5 mg/kg. Based on serum concentration measurements, the dosage regimen for individual patient was calculated by Sawchuk-Zaske's method. The calculated doses were compared to the prescribed doses in each patient. Of the patients on empirically prescribed doses 65% received 36% more drug than the calculated dose and 20% received 36% less than the calculated dose. The calculated dosing intervals were greater than the recommended intervals in 60% of the patients. The gentamicin trough concentration was greater than 2 micrograms/ml in 70% of the patients. There was a significant tendency to overdosage of the patients. For the kinetic group, following administration of the calculated dose, the steady-state peak and trough concentrations in each patient were measured. The correlation of measured to predicted steady-state serum concentrations was excellent (r = 0.9968, p less than 0.05). About 85% of the served trough concentrations and 90% of the peak values fell within the therapeutic range. The mean of the prediction error (ME), mean absolute error (MAE), mean squared error (MSE), and root mean squared error (RMSE) of the trough and peak concentrations were calculated. The 95% confidence interval of the ME for the trough and peak concentrations included zero, which shows that the prediction was not significantly biased.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed2005

Kinetic resolution and chemoenzymatic dynamic kinetic resolution of functionalized gamma-hydroxy amides.

[reaction: see text] An efficient kinetic resolution of racemic gamma-hydroxy amides 1 was performed via Pseudomas cepacia lipase (PS-C)-catalyzed transesterification. The enzyme PS-C tolerates both variation in the chain length and different functionalities giving good to high enantioselectivity (E values of up to >250). The combination of enzymatic kinetic resolution with a ruthenium-catalyzed racemization led to a dynamic kinetic resolution. The use of 2,4-dimethyl-3-pentanol as a hydrogen source to suppress ketone formation in the dynamic kinetic resolution yields the corresponding acetates in good yield and good to high enantioselectivity (ee's up to 98%). The synthetic utility of this procedure was illustrated by the practical synthesis of the versatile intermediate gamma-lactone (R)-5-methyltetrahydrofuran-2-one.

Journal Article↗

PubMed1997

Kinetics of dodecanedioic acid and effect of its administration on glucose kinetics in rats.

Dodecanedioic acid (C12), a saturated aliphatic dicarboxylic acid with twelve C atoms, was given as an intraperitoneal bolus to male Wistar rats, with the aim of evaluating C12 suitability as an energy substrate for parenteral nutrition. The 24 h urinary excretion of C12 was 3.9% of the administered dose. C12 kinetics were investigated by a one-compartment model with saturable tissue uptake and reversible binding to plasma albumin. The analysis of plasma concentration and urinary excretion data from different animals yielded the population means of the kinetic parameters: renal clearance was 0.72 ml/min per kg body weight (BW) (much smaller than inulin clearance in the rat), and maximal tissue uptake was 17.8 mumol/min per kg BW corresponding to 123.7 J/min per kg BW. These results encourage the consideration of C12 as a possible substrate for parenteral nutrition. To investigate the effect of C12 administration on glucose kinetics, two other groups of rats, one treated with an intraperitoneal bolus of C12 and the other with saline, were subsequently given an intravenous injection of D[-U-14C]glucose in a tracer amount. Radioactivity data of both control and C12-treated rats were analysed by means of a two-compartment kinetic model which takes into account glucose recycling. The estimates of glucose pool size (2.3 mmol/kg BW) and total-body rate of disappearance (82.1 mumol/min per kg BW) in control rats agreed with published values. In C12-treated rats, the rate of disappearance appeared to be reduced to 36.7 mumol/min per kg BW and the extent of recycling appeared to be negligible.

Animal Nutritional Physiological Phenomena↗

PubMed1970

Kinetics of C-14 Translocation in Soybean: II. Kinetics in the Leaf.

The kinetics of (14)C-assimilates in the soybean leaf were studied in pulse labeling and steady state labeling experiments. (14)C-Sucrose apparently served as the ultimate source, at least, of translocated (14)C-sucrose. However, since the specific activity of leaf sucrose reached a maximum within 5 minutes after pulse labeling, whereas that of exported sucrose did not reach a maximum until at least 20 minutes, it appeared that there were two sucrose compartments in the leaf. A possible physical basis for the two compartments may be the mesophyll (a photosynthetic compartment) and a specialized "paraveinal mesophyll" (a nonphotosynthetic compartment), through which photosynthate must pass on its way to the veins.The (14)C kinetics of sterol glucoside, and probably esterified sterol glucoside, were similar to those for (14)C-sucrose export. Sterol glucoside was labeled only in its glucose moiety and was the only stem lipid which became strongly labeled during (14)C-sucrose translocation. These sterol derivatives may act as membrane carriers of sucrose between the translocation stream and surrounding cells.The kinetics of (14)C-sucrose and its movement to the veins are discussed with reference to compartmentation within the leaf and metabolic exchange with other compounds, particularly with starch. Although a simple compartmental model gave a fairly accurate description of (14)C-sucrose kinetics, an entirely accurate model could not be provided, primarily because of loss of (14)C from sucrose, at an unknown rate, to starch.

Journal Article↗

PubMed1998

Inhibitors of glutathione reductase as potential antimalarial drugs. Kinetic cooperativity and effect of dimethyl sulphoxide on inhibition kinetics.

We have developed inhibitors of glutathione reductase that improve on the inhibition of literature lead compounds by up to three orders of magnitude. Thus, analogues of Safranine O and menadione were found to be strong, reversible inhibitors of yeast glutathione reductase. Safranine O exhibited partial, uncompetitive inhibition with Ki and alpha values of 0.5 mM and 0.15, respectively. Thionine O was a partial (hyperbolic) uncompetitive inhibitor with Ki and alpha values of 0.4 microM and 0.15, respectively. LY83583 and 2-anilino-1,4-naphthoquinone also showed (hyperbolic) partial, uncompetitive inhibition with micromolar Ki values. For Nile Blue A a model for two-site binding with (parabolic) uncompetitive inhibition fitted the data with a Ki value of 11 microM and a kinetic cooperativity between the sites of 0.12, increased to 0.46 by preincubation of the enzyme and Nile Blue A in the presence of glutathione disulphide. Analysis of the effects of preincubation on the kinetics and cooperativity indicated the possibility of a slow conformational change in the homodimeric enzyme, the first such indication of kinetic cooperativity in the native enzyme to our knowledge. Further evidence of conformational changes for this enzyme came from studies of the effects of dimethyl sulphoxide which indicated that this co-solvent, which at low concentrations has no apparent effect on initial velocities under normal assay conditions, induced a slow conformational change in the enzyme. Thionine O, Nile Blue A and LY83583 were redox-cycling substrates producing superoxide ion, detectable by means of cytochrome c reduction, but leading to no loss of glutathione reductase activity, under aerobic or anaerobic conditions. The water-soluble Safranine analogues Methylene Blue, Methylene Green, Nile Blue A and Thionine O (5 mg/kg i.p. x 5) were effective antimalarial agents in vivo against P. berghei, but their effect was small and a higher dose (50 mg/kg i.p. x 1) was toxic in mice. Comparison was made with human glutathione reductase and its literature-reported interactions with several tricyclic inhibitors as studied by X-ray diffraction. It is possible that the conformational changes detected in the present study from alterations in detailed kinetic inhibition mechanisms may shed light on information transfer through the glutathione reductase molecule from the dimer interface ligand pocket to the active-site.

Animals↗

PubMed1996

Relation among the resistance factor, kinetics of uptake, and kinetics of the P-glycoprotein-mediated efflux of doxorubicin, daunorubicin, 8-(S)-fluoroidarubicin, and idarubicin in multidrug-resistant K562 cells.

Multidrug resistance (MDR) is frequently associated with decreased cellular drug accumulation resulting from enhanced drug efflux. This is correlated with the presence of a membrane protein, the P-glycoprotein, which pumps a wide variety of drugs out of cells, reducing their intracellular concentration and thus their toxicity. The influx and efflux of drugs across the cell membrane are in large part responsible for their intracellular concentrations, and in the search for new compounds able to overcome MDR, it is of prime importance to determine the molecular parameters whose modification would lead to an increase in the kinetics of uptake and/or to a decrease in the P-glycoprotein-medicated efflux. Four anthracycline derivatives, doxorubicin, daunorubicin, 8-(S)-fluoroidarubicin, and idarubicin, which have the same amino sugar, were used to analyze the respective contribution of the kinetics of uptake and the P-glycoprotein-mediated efflux in their impaired accumulation in MDR cells. The kinetics of uptake of the four drugs vary over a very large range: the kinetics of uptake of daunorubicin, 8-(S)-fluoroidarubicin, and idarubicin are 16, 200, and 400 times higher than that of doxorubicin, respectively. However, the four drugs are extruded by P-glycoprotein at comparable rates. The apparent Km values for P-glycoprotein-mediated transport, the intracellular free cytosolic drug concentrations at half-maximal velocity for the cell lines used, were approximately 2.2 microM for daunorubicin and and approximately 1 microM for idarubicin and 8-(S)-fluoroidarubicin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

(S)-selective kinetic resolution and chemoenzymatic dynamic kinetic resolution of secondary alcohols.

(S)-Selective kinetic resolution was achieved through the use of a commercially available protease, which was activated with a combination of two different surfactants. The kinetic resolution (KR) process was optimized with respect to activation of the protease and to the acyl donor. The KR proved to be compatible with a range of functionalized sec-alcohols, giving good to high enantiomeric ratio values (up to >200). The enzymatic resolution was combined with a ruthenium-catalyzed racemization to give an (S)-selective dynamic kinetic resolution (DKR) of sec-alcohols. The DKR process works under very mild reaction conditions to give the corresponding esters in high yields and with excellent enantioselectivities.

Alcohols↗

PubMed1990

A microcalorimetric procedure for evaluating the kinetic parameters of enzyme-catalyzed reactions: kinetic measurements of the nitrogenase system.

The mechanism of nitrogenase catalysis, as evaluated from steady-state kinetic measurements, is presently unresolved primarily due to conflicting results regarding the reaction order of the nitrogenase reductant, S2O2-4, at high concentrations. A microcalorimetric method was developed and is described which measures the rate of heat production (and hence the rate of reactant disappearance or product formation) as a function of time. Because each substrate reaction order has a unique profile for the rate of heat production with time, the described procedure provides a means for establishing the substrate reaction order for the enzyme-catalyzed reaction under consideration by visual inspection of the resulting thermogram. The rate constant and other kinetic parameters are obtained from analysis of the shape of the thermogram and thermodynamic parameters are evaluated from either the shape of or the area bound by the thermogram. Application of this procedure to the nitrogenase system has confirmed one-half- and first-order reaction orders under limiting conditions for the S2O2-4 and MgATP substrates during the enzyme-catalyzed reaction for this important biological process. From a single thermogram, the enthalpy of reaction and the kinetic rate law are readily evaluated. The procedure is completely general in nature and is applicable to any chemical or biochemical system that evolves heat.

Azotobacter↗

PubMed1984

Kinetic studies of the reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: pH variation of kinetic parameters.

The specificity and kinetic parameters of the reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides has been examined under a range of conditions in order to elucidate details about the mechanism of action of this enzyme. The rate of oxidation of glucose 6-phosphate is inhibited by the addition of various organic solvents. However, the low, inherent glucose dehydrogenase activity of this enzyme was stimulated under these conditions, and was further activated by divalent anions that were observed to be inhibitors of the glucose 6-phosphate dehydrogenation. From an examination of the pH variation of the enzyme kinetic parameters two groups on the enzyme that appear to be involved in the binding of the phosphate group of the sugar substrate have been detected. An enzyme catalytic group, probably a carboxylic acid, has been identified that accepts the proton from the hydroxyl group at carbon-1 of the sugar substrate during its oxidation to a lactone. The ionization of a group on the enzyme with a pK of 8.7 resulted in an increase in the maximum velocity of the glucose-6-phosphate dehydrogenase activity of the enzyme as a consequence of a pH-dependent product release step that is no longer rate limiting at high pH. Stabilization of gluconic acid-delta-lactone against nonenzymatic hydrolysis by organic solvents has allowed the kinetic parameters of the reverse reaction to be reliably measured for the first time in a narrow pH range.

Anions↗

PubMed1985

Adenosinetriphosphate sulfurylase from Penicillium chrysogenum: steady-state kinetics of the forward and reverse reactions, alternative substrate kinetics, and equilibrium binding studies.

The kinetics of the forward ATP sulfurylase-catalyzed reaction were examined using a new assay based on 32PPi released from [gamma-32P]MgATP in the presence of inorganic sulfate. Replots yielded Vmaxf = 6.6 units mg protein-1, KmA = 0.13 mM, Kia = 0.33 mM, and KmB = 0.55 mM, where A = MgATP and B = SO2-4. Thiosulfate, a dead-end inhibitor of the reaction, was competitive with sulfate and noncompetitive with respect to MgATP. The ratio kcat/KmA was determined for several alternative inorganic substrates, B, where A = MgATP and B = SO2-4, SeO2-4, MoO2-4, WO2-4, or CrO2-4. For SO2-4 and SeO2-4, the ratio was 5-6.5 X 10(4) M-1 S-1; for the others, the ratio was 5.8-7.3 X 10(5) M-1 S-1. The results support a random addition of MgATP and inorganic substrate. The kinetics of the reverse reaction were examined using a new assay based on 35SO2-4 release from [35S]APS (adenosine 5'-phosphosulfate) in the presence of MgPPi. Reciprocal plots were linear, intersecting below the horizontal axis. Replots yielded Vmaxr = 50 units mg protein-1, KmQ = 0.3 microM, Kiq = 0.04 microM, and KmP = 4 microM, where Q = APS and P = PPi (total of all species). MgATP and SO2-4 were both competitive with APS and noncompetitive with respect to MgPPi. Taken together with earlier results suggesting that APS is competitive with both MgATP and SO2-4 and that MgPPi is noncompetitive with respect to both substrates, the qualitative results point to a random A-B, ordered P-Q kinetic mechanism. The Scatchard plot for [35S]APS binding was curved, indicating either negative cooperativity or more than a single class of sites. [gamma-32P]MgATP displayed half-site saturation in the presence of saturating FSO-3.

Adenosine Triphosphate↗