Effect of macroergic nucleotides and lysolecithin on the cholinergic reception of the heart muscle in the frog Rana temporaria

In experiments on isolated frog ventricle, it has been demonstrated that the dose-response curve for negative inotropic reaction of the myocardium to acetylcholine exhibits a sigmoid form, Hill's coefficient (nH) of this reaction being more than 1. The value of nH depends on the interval from isolation of the ventricle and on the duration of perfusion of the latter with Ringer's solution. It was shown that ATP, UTP, UDP and GTP in physiological concentrations induce both the increase in nH and the increase of K50 (acetylcholine concentration evoking the effect which is equal to half of the maximal one) of the investigated physiological reaction. Similar effects are produced by lysolecithin. Possible causes of "physiological cooperativity" of negative inotropic reaction of the myocardium to acetylcholine and the role of energy-rich nucleotides in this process are discussed.     

Effect of digitonin on the activity of Na,K-ATPase from bovine brain

The kinetic properties of intact and digitonin-treated Na,K-ATPase from bovine brain were studied. The temperature dependence curve for the rate of ATP hydrolysis under optimal conditions (upsilon 0) in the Arrhenius plots shows a break at 19-20 degrees. The temperature dependence curves for Km' and Km" have breaks at the same temperatures, while the Arrhenius plot for V is linear. The value of the Hill coefficient (nH) for ATP at 37 degrees is variable depending on ATP concentration, i. e. it is less than 1 at ATP concentrations below 50 mkM and is increased up to 3.2 at higher concentrations of the substrate. At high ATP concentrations the value of nH depends on temperature, falling down to 2.1 at 23 degrees and then down to 1 within the temperature range of 21-19 degrees. A further decrease in temperature does not significantly affect the nH value. Digitonin irreversibly inhibits Na, K-ATPase. ATP hydrolysis is more sensitive to the effect of the detergent than is nNPP hydrolysis, i. e. after complete inhibition of the ATPase about 40% of the phosphatase activity are retained. Treatment of Na,K-ATPase by digitonin results in elimination of the breaks in the Arrhenius plots for upsilon 0, Km' and Km", whereas the temperature dependence plot of V remains linear. Simultaneously digitonin eliminates the positive cooperativity of the enzyme for ATP. It is assumed that Na, K-ATPase from bovine brain is an oligomer of the (alpha beta) 4 type. Digitonin changes the type of interaction between the protomers within the oligomeric complex by changing the lipid environment of the enzyme or the type of protein -- lipid interactions.     

Kinetic cooperativity in the concerted model for allosteric enzymes.

The cooperativity of enzyme-substrate interactions is investigated in the concerted allosteric model of Monod, Wyman and Changeux. The general case of K-V systems is considered, in which the two protomer conformational states R and T postulated in the theory differ in catalytic and binding properties. An expression for the Hill coefficient nH defined with respect to the asymptotic velocity V infinity to is analyzed in conditions which exclude substrate inhibition. Kinetic cooperativity is always positive (nH greater than 1) in the case of a dimer enzyme, and in the case of an inactive T state. Slight kinetic negative cooperativity (nH less than 1) occurs under restrictive conditions for larger numbers of protomers when the substrate binds significantly to the less active state of the enzyme, but the phenomenon remains negligible for trimers and tetramers. These conclusions differ from those obtained [A. Goldbeter, J. Mol.Biol.90 (1974) 185] with the Hill coefficient based on the absolute maximum velocity, which may exceed the experimental value V infinity to in K-V systems. The results extend those of Paulus and DeRiel [J. Mol. Biol. 97 (1975) 667] and support the view that in most cases, negative cooperativity is not compatible with a mechanism based on a concerted and conservative allosteric transition. The Hill coefficients for binding and catalysis are compared in K-V systems.     

The control of adenylate cyclase by calcium in turkey erythrocyte ghosts.

The adenylate cyclase of turkey erythrocytes is inhibited by low concentrations of calcium. Calcium binds to the enzyme system so tightly that the enzyme can compete with ethylene glycol bis(beta-aminoethyl ether)-N, N1-tetraacetic acid (EGTA) for the metal. The calcium binding site is shown to be distinct from the magnesium binding sites required for activity. Thus Ca2+ functions as a negative allosteric effector. Calcium decreases dramatically the V max of the catecholamine-stimulated activity without affecting the affinity for the hormone or for the substrate ATP. The cooperativity in the response toward Mg2+ dependence (Hill coefficient, nH equals 3) is also unaffected by Ca2+ where as the S0.5 (concentration yielding one-half V max) for Mg2+ is affected only slightly. The Ca2+ effect is cooperative (nH equals 2) and therefore brought about by a cluster of Ca2+ binding sites. Mn2+ can substitute for Mg2+ as the enzyme activator but the Mn2+-activated enzyme is no longer inhibited by Ca2+. The possible physiological significance of the Ca2+ effect is discussed.     

Kinetic manifestations of slow isomerization of allosteric enzyme for the model of Monod, Wyman and Changeux]

A shape of the curves of a product accumulation in time (t) is analysed for the variant of Monod, Wyman and Changeux model which is characterized by comparable rates of equilibration between R and T enzyme forms on the one hand and the enzymatic process on the other hand. It is assumed that the complex of R and T forms with substrate are in rapid equilibrium with the free components. The character of the dependences of effective constant of R denoting T isomerization and the value of tau on substrate concentration are analysed (tau is the intercept of t-axis for linear asymptota of the curve of product concentration versus time at t leads to infinity). It is also shown that the low rate of R denoting T isomerization may be manifested by the shape of the plot of initial reaction rate versus substrate concentration unusual for the model of Monod et al. (the plots with intermediate plateau and ones with Hill's coefficient of cooperativity less than unity).     

Evaluation of Hill slopes and Hill coefficients when the saturation binding or velocity is not known.

1. The Hill coefficient (nH), an often-used measure of deviations from hyperbolic behaviour (nonhyperbolicity) in kinetic and binding systems, is usually estimated from the maximum or minimum slope of the Hill plot. The method depends strongly on the assumed magnitude of the asymptotic velocity (V) or binding (P) whose evaluation may be difficult in nonlinear/co-operative systems. Therefore, alternative procedures were devised for the estimation nH which do not require the prior knowledge of V or P. 2. When pairs of velocity/binding readings (v and w) are obtained at concentrations of c and alpha c, respectively (where alpha is a fixed constant), then the relation between w and v is described by a hyperbola, provided that Hill's equation is valid. In this case, linearizing plots, v/w versus v, w versus, w/v, and 1/w versus 1/v, can be used for estimation of the degree of the equation. However, if the Hill expression is applicable, these methods are not efficient and traditional procedures, particularly nonlinear regression, should be used. 3. The 'linearizing' plots of the Hill equation can be applied advantageously for the evaluation of the Hill slope and of nH also in the general case, when the Hill expression is actually not valid, provided that deviations from hyperbolic behaviour are positive. Appropriately extrapolated intercepts of the first two plots estimate alphanH. Furthermore, the slope of the third plot yields, similarly to the method of Kurganov et al., a continuous measure of the Hill slope (including its maximum) at all concentrations. The agreement is, at positive nonhyperbolicities, excellent theoretical values of Hill slopes and coefficients and those estimated by the proposed methods. 4. A coefficient of nonhyperbolicity (theta) is defined for 2nd-degree rate equations which provides a quantitative measure of positive or negative deviation from first-degree, hyperbolic characteristics. It is closely related to the Hill coefficient.     

Inhibition of beta-lactamase of Bacillus licheniformis 749/C by compound PS-5, a new beta-lactam antibiotic.

By use of a new computer-assisted u.v.-spectrophotometric assay method, the kinetic parameters of the reaction catalysed by Bacillus licheniformis 749/C beta-lactamase were re-examined and the mode of inhibition of the enzyme by compound PS-5, a novel beta-lactam antibiotic, was studied with benzylpenicillin as substrate. (1) The fundamental assay conditions for the determination of Km and V were examined in detail with benzylpenicillin as substrate. In 0.1 M-sodium/potassium phosphate buffer, pH 6.8, at 30 degrees C, initial substrate concentrations of benzylpenicillin above 0.7 mM were very likely to lead to substrate inhibition. The Km value of the enzyme for benzylpenicillin at initial concentrations from 1.96 to 0.07 mM was calculated to be 97-108 microM. (2) The Km values of the enzyme for 6-aminopenicillanic acid, ampicillin and cephaloridine were found to be 25, 154-161 and 144-161 microM respectively. (3) Compound PS-5 was virtually unattacked by Bacillus licheniformis 749/C beta-lactamase. (4) The activity of the enzyme was diminished by compound PS-5, to extents depending on the duration of incubation and the concentration of the inhibitor. The rate of inactivation of the enzyme by compound PS-5 followed first-order kinetics. (5) In an Appendix, a new computer-assisted u.v.-spectrophotometric enzyme assay method, in which a single reaction progress curve of time-absorbance was analysed by the integrated Michaelis-Menten equation, was devised for the accurate and precise determination of the kinetic constants of beta-lactamase. For conversion of absorbance readings into molar substrate concentrations, the initial or final absorbance reading that was independent of the reaction time was used as the basis of calculation. In calculation of Km and V three systematic methods of data combination were employed for finer analysis of the reaction progress curve. A list of the computer program named YF6TAIM is obtainable from the author on request or as Supplementary Publication SUP 50100 (12 pages) from the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., on the terms indicated in Biochem. J. (1978) 169, 5.     

The theoretical analysis of kinetic behaviour of “hysteretic” allosteric enzymes III. Dissociating and associating enzyme systems in which the rate of installation of equilibrium between the oligomeric forms is comparable to that of enzymatic reaction

The curves of the time (t) dependent product (Pr) accumulation for Monod, Wyman & Changeux model (1965), where the rate of installation of equilibrium between two conformational states of oligomeric enzyme (R T) is comparable to that of enzymatic process, are theoretically analysed. It is assumed that the complexes of R and T forms are in rapid equilibrium with the free components. The character of the effective rate constant of conformational transition R T and the value of τ (where τ is the intercept of the linear part of the kinetic curve of [Pr] versus t with the time axis) versus the substrate concentration is analysed. It is also shown that slow conformational transition R T can be manifested by an unusual shape for Monod et al. model plots of initial velocity of the enzyme reaction v. the substrate concentration (these curves can clearly display expressed inflection points and Hill's cooperativity coefficient less than unity).     

Dinucleoside polyphosphates stimulate the primer independent synthesis of poly(A) catalyzed by yeast poly(A) polymerase.

Novel properties of the primer independent synthesis of poly(A), catalyzed by the yeast poly(A) polymerase are presented. The commercial enzyme from yeast, in contrast to the enzyme from Escherichia coli, is unable to adenylate the 3'-OH end of nucleosides, nucleotides or dinucleoside polyphosphates (NpnN). In the presence of 0.05 mm ATP, dinucleotides (at 0.01 mm) activated the enzyme velocity in the following decreasing order: Gp4G, 100; Gp3G, 82; Ap6A, 61; Gp2G, 52; Ap4A, 51; Ap2A, 41; Gp5G, 36; Ap5A, 27; Ap3A, 20, where 100 represents a 10-fold activation in relation to a control without effector. The velocity of the enzyme towards its substrate ATP displayed sigmoidal kinetics with a Hill coefficient (nH) of 1.6 and a Km(S0.5) value of 0.308 +/- 0.120 mm. Dinucleoside polyphosphates did not affect the maximum velocity (Vmax) of the reaction, but did alter its nH and Km(S0.5) values. In the presence of 0.01 mm Gp4G or Ap4A the nH and Km(S0.5) values were (1.0 and 0.063 +/- 0.012 mm) and (0.8 and 0.170 +/- 0.025 mm), respectively. With these kinetic properties, a dinucleoside polyphosphate concentration as low as 1 micro m may have a noticeable activating effect on the synthesis of poly(A) by the enzyme. These findings together with previous publications from this laboratory point to a potential relationship between dinucleoside polyphosphates and enzymes catalyzing the synthesis and/or modification of DNA or RNA.     

Inhibition of the methylcoenzyme M methylreductase system by NAD+ and NADP+ in cell-extracts of Methanosarcina barkeri

In cell extracts of Methanosarcina barkeri, the methylcoenzyme M methylreductase system with H2 as the electron donor was inhibited by NAD+ and NADP+, but NADH and NADPH had no effect on enzyme activity. NAD+ (4 and 8 mM) shifted the saturation curve for methylcoenzyme M from hyperbolic (Hill coefficient [nH] = 1.0; concentration of substrate giving half maximal velocity [Km] = 0.21 mM) to sigmoidal (nH = 1.5 and 2.0), increased Km (Km = 0.25 and 0.34 mM), and slightly decreased Vmax. Similarly NADP+ at 4m and 8 mM increased nH to 1.6 and 1.85 respectively, but the Km values (0.3 and 0.56 mM) indicated that NADP+ was a more efficient inhibitor than NAD+.     

Effect of calixarene C-107 on kinetic parameters of Na+, K(+)-ATPase in the plasma membrane of the uterus myocytes

The inhibitory action of calixarene C-107 (5,17-diamino(2-pyridyl)methylphosphono- 11,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxy-calix[4]arene) on Na+, K(+)-ATPase activity kinetic properties of myometrium perforated plasma membrane was investigated. It has been shown that the calixarene C-107 inhibiting Na+, K(+)-ATPase does not change the kinetic parameters (Km, nH) of reaction velocity dependence on substrate concentration. The constant Ka of enzyme activation by MgCl2 has complex dependence on calixarene C-107 concentration: it increases twice with growth of calixarene concentration up to 50 nM and decreases to the control level with further growth of calixarene concentration. The Hill cooperativity coefficient nH of activation by MgCl2 does not vary in the presence of calixarene C-107. Both ATP and MgCl2 have no influence on Na+, K(+)-ATPase constant of inhibition by calixarene C-107, but an increase of concentration of the mentioned physiological compounds causes the growth of cooperativity coefficient nH of enzymatic reaction inhibition by calixaren C-107.     

AMP deaminase from baker's yeast. Purification and some regulatory properties.

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) was found in extract of baker's yeast (Saccharomyces cerevisiae), and was purified to electrophoretic homogeneity using phosphocellulose adsorption chromatography and affinity elution by ATP. The enzyme shows cooperative binding of AMP (Hill coefficient, nH, 1.7) with an s0.5 value of 2.6 mM in the absence or presence of alkali metals. ATP acts as a positive effector, lowering nH to 1.0 and s0.5 to 0.02 mM. P1 inhibits the enzyme in an allosteric manner: s0.5 and nH values increase with increase in Pi concentration. In the physiological range of adenylate energy charge in yeast cells (0.5 to 0.9), the AMP deaminase activity increases sharply with decreasing energy charge, and the decrease in the size of adenylate pool causes a marked decrease in the rate of the deaminase reaction. AMP deaminase may act as a part of the system that protects against wide excursions of energy charge and adenylate pool size in yeast cells. These suggestions, based on the properties of the enzyme observed in vitro, are consistent with the results of experiments on baker's yeast in vivo reported by other workers.     

The kinetics of heparin inhibition of the esterase and basal lipase activities of lipoprotein lipase

The kinetics of inhibition of the esterase and lipase activities of bovine milk lipoprotein lipase (LPL) were compared. The esterase LPL activity against emulsified tributyrylglycerol was not affected by the enzyme activator apolipoprotein C-II (C-II) and amounted to about 15% of the "plus activator" lipase enzyme activity. Heparin at concentrations of 20 micrograms/ml inhibited 25% of the esterase activity. The reaction followed Henri-Michaelis-Menten kinetics and the inhibition by heparin followed a linear, intersecting, noncompetitive kinetic model. On the other hand, the basal lipase activity of LPL against emulsified trioleoylglycerol (TG) was very sensitive to inhibition by heparin: 1 microgram/ml inhibited about 80% of the reaction and 3 micrograms/ml drove the reaction to zero. The velocity curve for the uninhibited basal LPL activity was sigmoidal with an apparent nH(TG) of 2.94. Heparin inhibited the lipase activity competitively: heparin decreased nH(TG) and increased[TG]0.5 6.4-fold, while TG decreased the nH(Heparin) from 2.14 to 0.95 and caused a 3-fold increase in [Heparin]0.5. C-II, at concentrations lower than 2.5 X 10(-8) M (i.e., lower than KA), countered the inhibitory effects of heparin: at constant inhibitor concentrations, C-II increased nH(TG) from 1.78 to 2.52 and decreased [TG]0.5 about 10-fold; it also increased the apparent Vmax. At the lower C-II concentrations, nH(C-II) was approximately equal to 1.0 and increasing the TG concentrations decreased [C-II]0.5 from 3.8 X 10(-8) to 8.5 X 10(-9) M, with no effect on the nH(C-II). At the higher C-II concentrations, nH(C-II) was 2.5 and TG decreased [C-II]0.5 about 2-fold with no effect on the nH(C-II). In the absence of heparin, C-II had no effect on nH(TG) nor on [TG]0.5, but it increased the apparent Vmax. On the other hand, TG had no effect on nH(C-II) nor on [C-II]0.5, but at any given C-II concentration, the reaction velocity increased with increasing TG concentrations. It is concluded that TG and heparin as well as C-II and heparin are mutually exclusive and that lipoprotein lipase is a multisite enzyme, possibly a tetramer, with three high-affinity catalytic sites, and an equal number of sites for C-II and heparin per oligomer. However, LPL differs from classical allosteric enzymes in that its activator has no effect on substrate cooperativity nor on [S]0.5; its only effect is to increase Vmax by increasing the catalytic rate constant kp by inducing conformational changes in the enzyme.     

Effects of monovalent cations on AMP nucleosidase from Azotobacter vinelandii.

The effect of monovalent cations on the purified AMP nucleosidase (AMP phosphoribohydrolase, EC 3.2.2.4) from Azotobacter vinelandii was investigated. All the monovalent cations were activators of the enzyme: Rb+ and Cs+ were the most effective, followed by K+, Na+, NH4+ and Li+ in that order. The apparent Ka for MgATP and nH values (Hill's interaction coefficient) decreased from 0.9 to 0.1 mM, and from 4 to 1, respectively, with the increase in K+ concentration, suggesting that the cation effects are on MgATP binding rather than catalysis. Gel filtration studies have revealed that the enzyme forms a non-dissociable enzyme species with a Stokes radius of 6.0--6.2 nm in the presence of saturating concentrations of monovalent cations, which can be distinguished from the 5.5-nm enzyme species showing temperature-dependent dissociation of the molecule in sulfate or phosphate. These results suggest that these ligands affect the association of the subunits through changes in the environment of the hydrophobic side chains of the enzyme molecules.     

Specific ligand-induced association of an enzyme. A new model of dissociating allosteric enzyme

The kinetic behavior of dissociative enzyme system of the type inactive monomer in equilibrium active dimer where dimeric form is stabilized by specific ligand (in particular by substrate) which is bound in the region of the contact of monomers has been analysed. It is assumed that the dissociation of dimer results in formation of monomers which retain the subsites for specific ligand binding. The shape of the dependences of enzyme reaction rate (v) on substrate concentration (S) has been characterized using the order of enzyme reaction rate with respect to substrate concentration: ns = d ln v/d ln [S]. When the substrate concentrations are low the dependences of v on [S] have S-shaped form (the maximum value of ns exceeds the unity) at the definite values of the parameters of the enzyme system. The value of ns approaches--2 at sufficiently high substrate concentrations (in the region where the substrate reveals the inhibitory effect due to blocking the association of inactive monomers into active dimer). The methods of calculation of the parameters of the dissociative enzyme system under discussion have been elaborated on the basis of the analysis of the experimental dependences of specific enzyme activity on enzyme concentration obtained at various fixed substrate concentrations.     

The oxidation of the highly tumorigenic benz[a]anthracene 3,4-dihydrodiol by rat liver dihydrodiol dehydrogenase

Rat liver dihydrodiol dehydrogenase (DDH, EC 1.3.1.20) has been shown to reduce the mutagenicity of benz[a]anthracene (BA) in the bacterial Ames test. BA-3,4-dihydrodiol is a highly mutagenic and tumorigenic metabolite of BA. In order to test the hypothesis that this dihydrodiol may be a substrate of DDH, we established two novel assay systems for the NADP(+)-dependent oxidation of BA-3,4-dihydrodiol by rat liver DDH, an HPLC-based assay procedure and a radiometric assay with specifically labelled [3,4-3H]-BA-3,4-dihydrodiol as substrate. With the HPLC-based assay, the kinetic constants of the enzymatic catalysis were as follows: Km(app) = 21 microM for BA-3,4-dihydrodiol and Vmax = 20.0 nmol/min.mg enzyme. The reaction product was identified by cochromatography, fluorimetry and mass spectroscopy as BA-3,4-catechol, but interconversions between the catechol and the corresponding o-quinone during the analytical procedures were detected. With the radiolabelled substrate, a linear relationship between substrate concentration and reaction velocity was found. The V/K value for labelled substrate was 0.155 ml/min.mg enzyme and a (V/K)H/(V/K)T kinetic isotope effect of 6.7 was observed. The non-labelled substrate acted as a competitive inhibitor of the enzymatic oxidation of tritiated BA-3,4-dihydrodiol with a Ki value of 56.4 microM. The reaction rates determined in this study suggest an important role of DDH activity in the metabolism of BA.     

A semi-integrated method for the determination of enzyme kinetic parameters and graphical representation of the Michaelis-Menten equation

A semi-integrated method for the determination of the enzyme kinetics parameters (Km and V) and graphical representation of the Michaelis-Menten equation is proposed as a variation of determination of initial reaction rate (v) as a function of initial substrate concentration ([S]0). The method is based on the determination of the time required to exhaust half of the initial substrate concentration as a function of the initial substrate concentration. The advantages and limitations of this method are discussed.     

Kinetic characterization of a T-state of Ascaris suum phosphofructokinase with heterotropic negative cooperativity by ATP eliminated.

The affinity analogue, 2',3'-dialdehyde ATP has been used to chemically modify the ATP-inhibitory site of Ascaris suum phosphofructokinase, thereby locking the enzyme into a less active T-state. This enzyme form has a maximum velocity that is 10% that of the native enzyme in the direction of fructose 6-phosphate (F6P) phosphorylation. The enzyme displays sigmoid saturation for the substrate fructose 6-phosphate (S0.5 (F6P) = 19 mM and nH = 2.2) at pH 6.8 and a hyperbolic saturation curve for MgATP with a Km identical to that for the native enzyme. The allosteric effectors, fructose 2,6-bisphosphate and AMP, do not affect the S0.5 for F6P but produce a slight (1.5- and 2-fold, respectively) V-type activation with Ka values (effector concentration required for half-maximal activation) of 0.40 and 0.24 mM, respectively. Their activating effects are additive and not synergistic. The kinetic mechanism for the modified enzyme is steady-state-ordered with MgATP as the first substrate and MgADP as the last product to be released from the enzyme surface. The decrease in V and V/K values for the reactants likely results from a decrease in the equilibrium constant for the isomerization of the E:MgATP binary complex, thus favoring an unisomerized form. The V and V/KF6P are pH dependent with similar pK values of about 7 on the acid side and 9.8 on the basic side. The microenvironment of the active site appears to be affected minimally as evidenced by the similarity of the pK values for the groups involved in the binding site for F6P in the modified and native enzymes.     

Studies on the kinetic effects of adenosine-3':5'-monophosphate-dependent phosphorylation of purified pig-liver pyruvate kinase type L.

The effect of cyclic-AMP-dependent phosphorylation on the activity of isolated pig liver pyruvate kinase was studied. It was found that the major kinetic effect of the phosphorylation was to reduce the affinity for the substrate phosphoenolpyruvate, K0.5 for this substrate increasing from 0.3 to 0.9 mM upon phosphorylation. The cooperative effect with phosphoenolpyruvate was enhanced, the Hill constant nH increasing concomitantly from 1.1 to 1.5. V was unaltered. The change in activity occurred in parallel with the phosphate incorporation, except during the initial part of the reaction, when inactivation was correspondingly slower. The affinity for the second substrate ADP was unchanged, with an apparent Km of 0.3 mM at saturating concentration of phosphoenolpyruvate. Likewise, the requirement for potassium was unaffected, whereas the phosphoenzyme required a higher concentration of magnesium ions for maximal activity, compared with the control enzyme. The inhibitory effect of the phosphorylation was counteracted by positive effectors, fructose 1,6-biphosphate in micromolar concentrations completely activated the phosphoenzyme, resulting in an enzyme with properties similar to the fructose 1,6-biphosphate-activated unphosphorylated enzyme, with K0.5 for phosphoenolpyruvate about 0.025 mM and with a Hill constant of 1.1. Hydrogen ions were also effective in activating the phosphoenzyme. Thus, when pH was lowered from 8 to 6.5 the inhibition due to phosphorylation was abolished. The phosphoenzyme was sensitive to further inhibition by negative effectors such as ATP and alanine. 2 mM ATP increased K0.5 for phosphoenolpyruvate to 1.5 mM and nH to 2.3. The corresponding values with alanine were 1.3 mM and 1.9. Phosphorylation is thought to be an additional mechanism of inhibition of the enzyme under gluconeogenetic conditions.     

Kinetics of soybean lipoxygenase reaction in hydrated reversed micelles

The rate of linoleic acid peroxidation catalysed by soybean lipoxygenase I was studied as a function of the hydration degree of aerosol OT (bis(2-ethylhexyl) sulfosuccinate sodium salt) reversed micelles in octane. Lipoxygenase reaction parameters for the micelle-bound substrate were spectrophotometrically determined. The linoleic acid distribution between the micelles and octane was detected by the sedimentation method, with the concentration of linoleic acid in supernatant after settling of micelles (i.e. the concentration of free linoleic acid) being estimated by the enzymatic method. The apparent constant of linoleic acid distribution (the ratio of the bound and free substrate concentrations) was enhanced with increasing hydration of reversed micelles. The dependence of the enzymatic reaction rate on the bound substrate concentration obeyed the empiric Hill equation. The Hill coefficient remained practically constant (h = 1.34) as the hydration degree changed. Parameters of the lipoxygenase reaction, enzyme reaction limiting rate V and semi-saturation substrate concentration [S]0.5 increased with increasing degree of hydration and reached the optimum at [H2O]/[AOT] approximately 30, where dimensions of the micellar internal cavity coincided with those of the enzyme molecule. Some aspects of kinetic behavior of membrane-bound enzymes participating in chemical transformation of non-polar compounds dispersed in lipid phase are discussed.