Cooperative effects of CTP on calf liver CTP synthetase.

In all previous kinetics studies of calf liver CTP synthetase, simple Michaelis-Menten hyperbolic plots were obtained. In this study it was shown that calf liver CTP synthetase could generate sigmoidal kinetic plots as a function of the substrate UTP when in the presence of the product of the reaction, CTP. The Hill number was estimated to be 2.8. The enzyme did not generate sigmoidal plots as a function of the other substrates (L-glutamine and ATP) either in the presence or absence of CTP. Thus, CTP apparently induced changes in the liver enzyme which altered the binding of UTP to the enzyme by acting at a site distinct from the UTP binding site (allosteric site). This concept was further strengthened by the fact that 3-deazaUTP, a known competitive inhibitor of the liver enzyme, did not induce sigmoidal kinetic plots. It was also shown that CTP had no effect upon the dimerization of the enzyme, thus ruling out monomer to dimer transitions as a potential mechanism for the observed sigmoidal kinetics.     

Activation of D-tyrosine by Bacillus stearothermophilus tyrosyl-tRNA synthetase: 2. Cooperative binding of ATP is limited to the initial turnover of the enzyme

The activation of D-tyrosine by tyrosyl-tRNA synthetase has been investigated using single and multiple turnover kinetic methods. In the presence of saturating concentrations of D-tyrosine, the activation reaction displays sigmoidal kinetics with respect to ATP concentration under single turnover conditions. In contrast, when the kinetics for the activation reaction are monitored using a steady-state (multiple turnover) pyrophosphate exchange assay, Michaelis-Menten kinetics are observed. Previous investigations indicated that activation of l-tyrosine by the K233A variant of Bacillus stearothermophilus tyrosyl-tRNA synthetase displays sigmoidal kinetics similar to those observed for activation of d-tyrosine by the wild-type enzyme. Kinetic analyses indicate that the sigmoidal behavior of the d-tyrosine activation reaction is not enhanced when Lys-233 is replaced by alanine. This supports the hypothesis that the mechanistic basis for the sigmoidal behavior is the same for both d-tyrosine activation by wild-type tyrosyl-tRNA synthetase and activation of l-tyrosine by the K233A variant. The observed sigmoidal behavior presents a paradox, as tyrosyl-tRNA synthetase displays an extreme form of negative cooperativity, known as "half-of-the-sites reactivity," with respect to tyrosine binding and tyrosyl-adenylate formation. We propose that the binding of D-tyrosine weakens the affinity with which ATP binds to the functional subunit in tyrosyl-tRNA synthetase. This allows ATP to bind initially to the nonfunctional subunit, inducing a conformational change in the enzyme that enhances the affinity of the functional subunit for ATP. The observation that sigmoidal kinetics are observed only under single turnover conditions suggests that this conformational change is stable over multiple rounds of catalysis.     

Role of substrate inhibition kinetics in enzymatic chemical oscillations.

Two chemical kinetic models are investigated using standard nonlinear dynamics techniques to determine the conditions under which substrate inhibition kinetics can lead to oscillations. The first model is a classical substrate inhibition scheme based on Michaelis-Menten kinetics and involves a single substrate. Only when this reaction takes place in a flow reactor (i.e., both substrate and product are taken to follow reversible flow terms) are oscillations observed; however, the range of parameter values over which such oscillations occur is so narrow it is experimentally unobservable. A second model based on a general mechanism applied to the kinetics of many pH-dependent enzymes is also studied. This second model includes both substrate inhibition kinetics as well as autocatalysis through the activation of the enzyme by hydrogen ion. We find that it is the autocatalysis that is always responsible for oscillatory behavior in this scheme. The substrate inhibition terms affect the steady-state behavior but do not lead to oscillations unless product inhibition or multiple substrates are present; this is a general conclusion we can draw from our studies of both the classical substrate inhibition scheme and the pH-dependent enzyme mechanism. Finally, an analysis of the nullclines for these two models allows us to prove that the nullcline slopes must have a negative value for oscillatory behavior to exist; this proof can explain our results. From our analysis, we conclude with a brief discussion of other enzymes that might be expected to produce oscillatory behavior based on a pH-dependent substrate inhibition mechanism.     

Biological processes in organised media

Embedding a simple Michaelis-Menten enzyme in a gel slice may allow the catalysis of not only scalar processes but also vectorial ones, including uphill transport of a substrate between two compartments, and may make it seem as if two enzymes or transporters are present or as if an allosterically controlled enzyme/transporter is operating. The values of kinetic parameters of an enzyme in a partially hydrophobic environment are usually different from those actually measured in a homogeneous aqueous solution. This implies that fitting kinetic data (expressed in reciprocal co-ordinates) from in vivo studies of enzymes or transporters to two straight lines or a sigmoidal curve does not prove the existence of two different membrane mechanisms or allosteric control. In the artificial transport systems described here, a functional asymmetry was sufficient to induce uphill transport, therefore, although the active transport systems characterised so far correspond to proteins asymmetrically anchored in a membrane, the past or present existence of structurally symmetrical systems of transport in vivo cannot be excluded. The fact that oscillations can be induced in studies of the maintenance of the electrical potential of frog skin by addition of lithium allowed evaluation of several parameters fundamental to the functioning of the system in vivo (e.g., relative volumes of internal compartments, characteristic times of ionic exchanges between compartments). Hence, under conditions that approach real biological complexity, increasing the complexity of the behaviour of the system may provide information that cannot be obtained by a conventional, reductionist approach.     

Analysis of a novel diacylglycerol kinase from Dictyostelium discoideum: DGKA

Diacylglycerol kinases (DGKs) catalyze the ATP-dependent phosphorylation of diacylglycerols to generate phosphatidic acid and have been investigated in prokaryotic and eukaryotic organisms. Recently, a protein that is significantly similar to human DGK-theta, DGKA, was identified in Dictyostelium discoideum. It has been shown to possess DGK activity when assayed using a medium-chain diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DiC8). A complete understanding of DGK catalytic and regulatory mechanisms, as well as physiological roles, requires an understanding of its biochemical and kinetic properties. This report presents an analysis of these properties for DGKA. The enzyme catalyzes the phosphorylation of DiC8, and another medium-chain DAG, DiC6 (1,2-dihexanoyl-sn-glycerol), in a Michaelis-Menten manner. Interestingly, the kinetics of DGKA using physiologically relevant long-chain DAGs was dependent on substrate surface concentration and the detergent that was used. DGKA displayed Michaelis-Menten kinetics with respect to bulk substrate concentration (1,2-dioleoyl-sn-glycerol) in octyl glucoside mixed micelles when the surface substrate concentration was at or below 3.5 mol %. At higher surface concentrations, however, there was a sigmoidal relationship between the initial velocity and bulk substrate concentration. In contrast, DGKA displayed sigmoidal kinetics with respect to bulk substrate concentrations at all surface concentrations in Triton X-100 mixed micelles. Finally, we show the catalytic activity of DGKA was significantly enhanced by phosphatidylserine (PS) and phosphatidic acid (PA).     

A single nucleotide polymorphism of CYP2b6 found in Japanese enhances catalytic activity by autoactivation

A single nucleotide polymorphism (SNP) resulting in a substitution from Gln to His was found in exon 4 of the CYP2B6 gene in Japanese. The frequency of the variant allele was found to be 19.9%. The mutant- and the wild-type enzymes were expressed in Escherichia coli, and the effects of the single amino acid substitution on the catalytic activity were examined by investigating the kinetic profiles of 7-ethoxycoumarin O-deethylase activity. The wild-type enzyme showed typical Michaelis-Menten kinetics, while the mutant-type enzyme represented the sigmoidal kinetics with a higher V(max) value compared to that of the wild-type enzyme. Eadie-Hofstee plots further revealed an existence of allosteric effects for the reaction catalyzed by the variant. This is the first evidence demonstrating that only one amino acid substitution, Gln172His, caused by natural SNP enhances the catalytic activity of CYP by obtaining the character of homotropic cooperativity.     

Purification and characterization of pyruvate decarboxylase from Sarcina ventriculi.

Pyruvate decarboxylase from the obligate anaerobe Sarcina ventriculi was purified eightfold. The subunit Mr was 57,000 +/- 3000 as estimated from SDS-PAGE, and the native Mr estimated by gel filtration on a Superose 6 column was 240,000, indicating that the enzyme is a tetramer. The Mr values are comparable to those for pyruvate decarboxylase from Zymomonas mobilis and Saccharomyces cerevisiae, which are also tetrameric enzymes. The enzyme was oxygen stable, and had a pH optimum within the range 6.3-6.7. It displayed sigmoidal kinetics for pyruvate, with a S0.5 of 13 mM, kinetic properties also found for pyruvate decarboxylase from yeast and differing from the Michaelis-Menten kinetics of the enzyme from Z. mobilis. No activators were found. p-Chloromercuribenzoate inhibited activity and the inhibition was reversed by the addition of dithiothreitol, indicating that cysteine is important in the active site. The N-terminal amino acid sequence of pyruvate decarboxylase was more similar to the sequence of S. cerevisiae than Z. mobilis pyruvate decarboxylase.     

The Salt Absorption Isotherm

It is shown that the absorption isotherm of rubidium by excised barley roots can be explained either by two uptake mechanisms following Michaelis-Menten kinetics or by two mechanisms, one actively transporting salts into the tissue (the pump), the other one being more passive in nature (the leak), operating in either direction, depending on external and internal substrate concentration. Kinetic data are thus consistent with more than one transport model. It was further demonstrated for the pair K-Na, that a competitor not only reduces salt uptake but can also reverse the direction of net flux. This observation cannot be explained by classical enzyme kinetics, it is, however, consistent with the pump and leak system. Just as Michaelis-Menten kinetics, the pump and leak system can explain ion competition, in addition it offers a possible explanation of the Viets' effect and it can explain the time curve of absorption.     

Lipase-catalysed hydrolysis of short-chain substrates in solution and in emulsion: a kinetic study.

We have studied the enzymatic hydrolysis of solutions and emulsions of vinyl propionate, vinyl butyrate and tripropionin by lipases of various origin and specificity. Kinetic studies of the hydrolysis of short-chain substrates by microbial triacylglycerol lipases from Rhizopus oryzae, Mucor miehei, Candida rugosa, Candida antarctica A and by (phospho)lipase from guinea-pig pancreas show that these lipolytic enzymes follow the Michaelis-Menten model. Surprisingly, the activity against solutions of tripropionin and vinyl esters ranges from 70% to 90% of that determined against emulsions. In contrast, a non-hyperbolic (sigmoidal) dependence of enzyme activity on ester concentration is found with human pancreatic lipase, triacylglycerol lipase from Humicola lanuginosa (Thermomyces lanuginosa) and partial acylglycerol lipase from Penicillium camembertii and the same substrates. In all cases, no abrupt jump in activity (interfacial activation) is observed at substrate concentration corresponding to the solubility limit of the esters. Maximal lipolytic activity is always obtained in the presence of emulsified ester. Despite progress in the understanding of structure-function of lipases, interpretation of the mode of action of lipases active against solutions of short-chain substrates remains difficult. Actually, it is not known whether these enzymes, which possess a lid structure, are in open or/and closed conformation in the bulk phase and whether the opening of the lid that gives access to the catalytic triad is triggered by interaction of the enzyme molecule with monomeric substrates or/and multimolecular aggregates (micelles) both present in the bulk phase. From the comparison of the behaviour of lipases used in this study which, in some cases, follow the Michaelis-Menten model and, in others, deviate from classical kinetics, it appears that the activity of classical lipases against soluble short-chain vinyl esters and tripropionin depends not only on specific interaction with single substrate molecules at the catalytic site of the enzyme but also on physico-chemical parameters related to the state of association of the substrate dispersed in the aqueous phase. It is assumed that the interaction of lipase with soluble multimolecular aggregates of tripropionin or short-chain vinyl esters or the formation of enzyme-substrate mixed micelles with ester bound to lipase, might represent a crucial step that triggers the structural transition to the open enzyme conformation by displacement of the lid.     

Lipoxygenase activity in the brain regions of young chicks: isolation and some properties.

1. The lipoxygenase (LOX) oxygenation pathway of arachidonic acid was investigated in the cerebellum and cerebral hemispheres of young chicks. 2. Lipoxygenase products consisted mainly of 15-hydroxyeicosatetraenoic acid (15-HETE), accompanied by the 15-hydroperoxy analog (15-HPETE) and the 5-HETE product. 3. The yield of 15-HETE was 3 times greater in the cerebellar system than in the cerebrum. 4. PLA2 activity of the cerebellum was twice that of the cerebrum. 5. Affinity chromatography revealed 2 brain fractions with LOX activity which were assayed with either linoleic or arachidonic acid as substrate. 6. The fraction eluted with 0.2 M sodium acetate pH 5.0, produced a higher yield and enrichment of LOX activity than the eluate obtained with 0.1 M Tris-HCl buffer (pH 8.0). 7. A considerably higher yield and enrichment of the enzyme was achieved when the starting material was the cerebellum, compared to the cerebrum. 8. The optimal pH for both purified fractions from cerebrum and cerebellum was 6.5, with either linoleic or arachidonic acid as substrate. 9. The cerebral LOX yielded Michaelis-Menten kinetics when linoleic acid was the substrate, while the corresponding plots for the cerebellar enzyme were sigmoidal. 10. Arachidonic acid as substrate produced sigmoidal plots, except at pH 5.0, where Michaelis-Menten kinetics were observed. 11. These results and the elevated activities of PLA2 and 15-LOX could be significant in relation to the special vulnerability of the cerebellum in chick nutritional encephalomalacia.     

A simple computer program with statistical tests for the analysis of enzyme kinetics.

A simple computer program that calculates the kinetic parameters of enzyme reactions is described. Parameters are determined by nonlinear, least-squares regression using either Marquardt-Levenberg or Gauss-Newton algorithms to find the minimum sum of squares. Three types of enzyme reactions can be analyzed: single substrate reactions (Michaelis-Menten and sigmoidal kinetics), enzyme activation at a fixed substrate value or enzyme inhibition at a fixed substrate value. The user can monitor goodness of fit through nonparametric statistical tests (performed automatically by the computer) and through visual examination of the pattern of residuals. The program is unique in providing equations for activator and inhibition analysis as well as in enabling the user to fix some of the parameters before regression analysis. The simplicity of the program makes it extremely useful for quickly determining kinetic parameters during the data-gathering process.     

Stabilization of active form of rabbit liver phosphofructokinase

(1) The active form of rabbit liver phosphofructokinase when preincubated in presence of F^? and effectors of the enzyme is stabilized against its conversion to less active form as a result of dilution. (2) The stabilized active form of enzyme has a Km value of 0.01 mM for fructose 6-phosphate, the same as measured in presence of all the positive effectors, and is lower, by 13 times, than the Km value of the non-stabilized control enzyme, and exhibits normal Michaelis-Menten kinetics, in contrast to the non-stabilized control enzyme which shows sigmoidal kinetics. (3) The stabilized active form of enzyme is neither inhibited by excess concentration of ATP nor activated by activators of phosphofructokinase. (4) The data thus support the proposition that the enzyme does indeed exist in two interconvertible forms with enormous difference in their affinities for fructose 6-phosphate and effectors.     

Investigations of reaction kinetics for immobilized enzymes--identification of parameters in the presence of diffusion limitation

A method is proposed for identification of kinetic parameters when diffusion of substrates is limiting in reactions catalyzed by immobilized enzymes. This method overcomes conventional sequential procedures, which assume immobilization does not affect the conformation of the enzyme and, thus, consider intrinsic and inherent kinetics to be the same. The coupled equations describing intraparticle mass transport are solved simultaneously using numerical methods and are used for direct estimation of kinetic parameters by fitting modeling results to time-course measurements in a stirred tank reactor. While most traditional procedures were based on Michaelis-Menten kinetics, the method presented here is applicable to more complex kinetic mechanisms involving multiple state variables, such as ping-pong bi-bi. The method is applied to the kinetic resolution of (R/S)-1-methoxy-2-propanol with vinyl acetate catalyzed by Candida antarctica lipase B. A mathematical model is developed consisting of irreversible ping-pong bi-bi kinetics, including competitive inhibition of both enantiomers. The kinetic model, which fits to experimental data over a wide range of both substrates (5-95%) and temperatures (5-56 degrees C), is used for simulations to study typical behavior of immobilized enzyme systems.     

Transient response of encapsulated enzymes in hollow-fiber reactor

A mathematical model for the transient response of encapsulated enzymes is developed showing the effects of the outer boundary layer, the encapsulating membrane, the partition coefficient, and diffusion with reaction within the encapsulating medium. The model incorporates both first-order kinetics and Michaelis-Menten kinetics for the reaction rate. Using typical hollow-fiber or microcapsule parameters, the model shows that (a) the partition coefficient affects the overall rate only when the rate-limiting step is diffusion through the membrane, (b) the transient overall effectiveness factor rises sharply with time and approaches an asymptotic value for most situations, and (c) the first-order approximation to Michaelis-Menten kinetics is not valid when the initial outside bulk concentration is higher than the Michaelis constant and the overall rate is reaction limited. The model is compared with experimental data using uricase in a hollow-fiber enzyme reactor configuration. Batch assay and CSTUER (continuous-stirred ultrafiltration enzyme reactor) studies were conducted on the free enzyme to provide some of the parameters used in the model. The CSTUER data fit the case of substrate inhibition kinetics with the apparent Michaelis constant approaching zero. The hollow-fiber reactor was conducted with uricase dissolved in both a buffer solution and a concentrated hemoglobin solution. Diffusivities of the solute were measured in both solutions as was the osmotic pressure of the hemoglobin solution. While experimental data for uricase in buffer solution could easily be matched by the model, that in the concentrated hemoglobin solution could not.     

Steady state kinetics of an enzyme reaction with one substrate and one modifier

This study examines the steady state kinetics of a reaction involving an enzyme, a substrate and a modifier when the reaction follows Michaelis-Menten kinetics. Conditions for Michaelis-Menten kinetics are deduced, and it is shown that an analogue of detailed balance determines the complexity of the rate equations in these cases. A scheme to distinguish many cases of Michaelis-Menten kinetics is presented. It is shown that steady state kinetics are, in general, insufficient to specify the mechanism of a reaction, since different effects of a modifier can give identical steady state kinetic data.     

Alpha-fucosidase-ganglioside interactions. Action of alpha-L-fucosidase from the hepatopancreas of Octopus vulgaris on a fucose-containing ganglioside (Fuc-GM1).

alpha-L-Fucosidase, prepared in highly purified form (Mr 70 000-74 000) from Octopus hepatopancreas, was able to hydrolyse a fucose-containing ganglioside, namely Fuc-GM1 (II3NeuAc,IV2Fuc-GgOse4-Cer). The enzyme showed an irregular kinetic behaviour (v/[S] and v/[E] relationships following sigmoidal curves) when working on micellar Fuc-GM1 (Mr of the micelle 500 000), but obeyed regular hyperbolic kinetics when acting on low-Mr substances. It was observed that, on incubation with micellar Fuc-GM1 under the conditions used for the enzyme assay, Octopus alpha-L-fucosidase produced a ganglioside-enzyme complex that was catalytically inactive. This complex had an Mr exceeding 500 000 and a ganglioside/protein ratio of 4:1 (w/w), which is consistent with a stoichiometric combination of one ganglioside micelle with two enzyme molecules. Inactivation of alpha-L-fucosidase by formation of the corresponding complexes was also obtained with micellar gangliosides GM1 (II3NeuAc-GgOse4-Cer), GD1a (II3NeuAc,IV3NeuAc-GgOse4-Cer) and GT1b [II3(NeuAc)2,IV3-NeuAc-GgOse4-Cer], which are not substrates for the enzyme, indicating that the ganglioside micelles per se act as enzyme inhibitors. However, alpha-L-fucosidase easily forms a Fuc-GM1-alpha-L-fucosidase complex, displaying regular Michaelis-Menten kinetics. Therefore the anomalous behaviour exhibited by alpha-L-fucosidase on micellar Fuc-GM1 is likely due to formation of the complex, which separates the fucosyl linkage from the active site of the complexed enzyme, but makes it available to the enzyme in the free form.     

A cellular automata model of enzyme kinetics

We have developed a cellular automata model of an enzyme reaction with a substrate in water. The model produces Michaelis-Menten kinetics with good Lineweaver-Burk plots. The variation in affinity parameters predicts that, in general, hydrophobic substrates are more reactive with enzymes, this attribute being more important than the relationship between enzyme and substrate. The ease of generation and the illustrative value of the model lead us to believe that cellular automata models have a useful role in the study of dynamic phenomena such as enzyme kinetics.     

Regulation of pyruvate kinases from Fusarium oxysporum.

Two types of pyruvate kinases were found in Fusarium oxysporum. One type (inducible) was present mainly during the early stages of growth on glucose or sucrose and displayed Michaelis-Menten kinetics with respect to phosphoenolpyruvate and adenosine diphosphate. The major type (constitutive) was present under all conditions of growth and displayed in the absence of potassium ions, a sigmoidal substrate saturation curve when phosphoenolpyruvate was used as the variable substrate. In the presence of potassium ions the saturation curve for phosphoenolpyruvate exhibits a plateau at half-maximal velocity. The effects of various metabolites on the activity of the inducible and constitutive kinases were also studied. Fructose-1,6-diphosphate, cyclic AMP, acetyl CoA, tryptophan, and phenylalanine had no effect on the activity of the enzymes. Citrate was a potent inhibitor of the constitutive pyruvate kinase activity and increased the sigmoidicity of the saturation curve for phosphoenolpyruvic acid. In the presence of K+, the bimodal plot observed in the absence of citrate gradually changed to a hyperbolic shape as the concentration of citric acid was increased. In the presence of K+ and ADP as the variable substrate citric acid converted the hyperbolic plot to a sigmoidal one. Citrate had no effect on the inducible enzyme.     

Calculating the probability of detection for inhibitors in enzymatic or binding reactions in high-throughput screening

In high-throughput screening (HTS) for drug candidates from a library containing tens of thousands to millions of chemical compounds, one problem is assessing the sensitivity of an assay for detecting compounds with a particular potency. For example, when looking for inhibitors of an enzyme, what is the potency of an inhibitor that will be readily detected by an enzyme inhibition assay? Similarly, when assessing compounds that inhibit binding between receptors and ligands or similar molecule-to-molecule interactions, what potency of an inhibitor will be readily detected? In this article, the well-established concepts of Michaelis-Menten kinetics and Langmuir binding isotherms are combined with fundamental statistical principles to yield a measure of assay sensitivity. The approach is general and can be modified to accommodate situations where the reaction kinetics is known to be more complicated than situations described by the Michaelis-Menten and Langmuir equations. The calculations presented take into account the concentration of inhibitor used, the variability of the assay, the relationship between the K(m) or K(d) of the reaction and the substrate or ligand concentration used, the threshold or cutoff value used for determining "hits," and the number of replicates used in screening.