An enzyme electrode for specific determination of L-lysine: A real-time control sensor

Lysine decarboxylase (L-lysine carboxylyase, E.C.4.1.1.18) is immobolized on a carbon dioxide gas-sensing electrode, by copolymerization with gelatin using the bifuncitional agent glutaraldehyde. The enzyme electrodes thus prepared are used in a continuous flow system to measure the concentration of L-lysine in a mixture of amino acids. The measuring time for each sample is about 3 min, including response and rinsing times. The electrode response is linear between 0.01-1 g/L and has a high specificity for L-lysine. The enzyme electrode response to lysine at concentrations below 0.5 g/L is stable on repeated use for at least 500 assays.     

Enzyme electrode for specific determination of L-lysine

L-Lysine alpha-oxidase from Trichoderma viride Y244-2 is immobilized in a gelatin support and fixed on a pO(2) sensor. The enzyme electrode obtained is used in a continuous flow system in order to measure the concentration of L-lysine in a fermentor. The sample oxygen-content dependance of the signal is minimized because of the enzyme support properties. The enzyme electrode response is set for lysine concentration from 0.2mM to 4mM. The specificity of lysine is tested with other amino acids. The enzyme membrane for lysine electrode can be used 3000 times or stored six months with good stability.     

A method to describe enzyme-catalyzed reactions by combining steady state and time course enzyme kinetic parameters

Background

Complete analysis of single substrate enzyme-catalyzed reactions has required a separate use of two distinct approaches. Steady state approximations are employed to obtain substrate affinity and initial velocity information. Alternatively, first order exponential decay models permit simulation of the time course data for the reactions. Attempts to use integrals of steady state equations to describe reaction time courses have so far met with little success.

Methods

Here we use equations based on steady state approximations to directly model time course plots.

Results

Testing these expressions with the enzyme β-galactosidase, which adheres to classical Michaelis–Menten kinetics, produced a good fit between observed and calculated values.

General significance

This study indicates that, in addition to providing information on initial kinetic parameters, steady state approximations can be employed to directly model time course kinetics.Integrated forms of the Michaelis–Menten equation have previously been reported in the literature. Here we describe a method to directly apply steady state approximations to time course analysis for predicting product formation and simultaneously obtain multiple kinetic parameters.     

Acetyl coenzyme A carboxylase. Rapid purification of the chick liver enzyme and steady state kinetic analysis of the carboxylase-catalyzed reaction

Avidin affinity chromatography was used to rapidly purify acetyl-CoA carboxylase to homogeneity in high yield from chicken liver. Dissociation of the purified carboxylase with dodecyl sulfate yielded a single size class of subunit polypeptide of 225,000 daltons. A steady state kinetic analysis of the carboxylase-catalyzed carboxylation of acetyl-CoA gave rise to intersecting line patterns in all double-reciprocal plots of initial velocity with each substrate pair, i.e. ATP . Mg and HCO3(-) and acetyl-CoA. It was concluded that the kinetic mechanism involves a quaternary complex of the enzyme, ADP, Pi, and acetyl-CoA rather than a double displacement as previously believed. The ordered addition of ATP, HCO3(-), and then acetyl-CoA, to the citrate-activated form of the carboxylase is the kinetic mechanism most consistent with the results.     

Continuous measurement of ethanol production by aerobic yeast suspensions with an enzyme electrode

Summary An alcohol electrode was constructed which consisted of an oxygen probe onto which alcohol oxidase was immobilized. This enzyme electrode was used, in combination with a reference oxygen electrode, to study the short-term kinetics of alcoholic fermentation by aerobic yeast suspensions after pulsing with glucose. The results demonstrate that this device is an excellent tool in obtaining quantitative data on the short-term expression of the Crabtree effect in yeasts.Samples from aerobic glucose-limited chemostat cultures of Saccharomyces cerevisiae not producing ethanol, immediately (within 2 min) exhibited aerobic alcoholic fermentation after being pulsed with excess glucose. With chemostat-grown Candida utilis, however, ethanol production was not detectable even at high sugar concentrations. The Crabtree effect in S. cerevisiae was studied in more detail with commercial baker's yeast. Ethanol formation occurred only at initial glucose concentrations exceeding 150 mg·l^-1, and the rate of alcoholic fermentation increased with increasing glucose concentrations up to 1,000 mg·l^-1 glucose.Similar experiments with batch cultures of certain non-fermentative yeasts revealed that these organisms are capable of alcoholic fermentation. Thus, even under fully aerobic conditions, Hansenula nonfermentans and Candida buffonii produced ethanol after being pulsed with glucose. In C. buffonii ethanol formation was already apparent at very low glucose concentrations (10 mg·l^-1) and alcoholic fermentation even proceeded at a higher rate than in S. cerevisiae. With Rhodotorula rubra, however, the rate of ethanol formation was below the detection limit, i.e., less than 0.1 mmol·g cells^-1·h^-1.     

Rapid determination of kinetic constants by competitive spectrophotometry

The use of competitive spectrophotometry to measure kinetic constants for enzyme-catalyzed reactions is described. The equation for the progress curve characterizing the kinetic behavior of an enzyme acting simultaneously on two alternative substrates is derived. By the addition of a competition term to the integrated Michaelis-Menten equation, the kinetic constants of an alternative substrate can be evaluated by measuring the competition with a substrate of known kinetic constants in a single experiment. Studies are presented involving the enzymes leucine aminopeptidase (LAP) and carboxypeptidase A (CPA). The results obtained with LAP and CPA showed that the kinetic constants determined using competitive spectrophotometry were in agreement with values cited in the literature or with values determined by single substrate enzyme kinetics.     

Fatty acid synthetase. A steady state kinetic analysis of the reaction catalyzed by the enzyme from pigeon liver.

The kinetic mechanism of pigeon liver fatty acid synthetase action has been studied using steady state kinetic analysis. Initial velocity studies are consistent with an earlier suggestion that the enzyme catalyzes this reaction by a seven-site ping-pong mechanism. Although the range of substrate concentrations that could be used was limited by several factors, the initial velocity patterns showing the relationship between the substrates acetyl coenzyme CoA, malonyl-CoA, and NADPH appear to be a series of parallel lines, regardless of which substrate is varied at fixed levels of a second substrate. However, two of the substrates, acetyl-CoA and malonly-CoA, apparently exhibit a competitive substrate inhibition with respect to each other, but NADPH shows no inhibition of any kind. Product inhibition patterns suggest that free CoA is competitive versus acetyl-CoA and malonyl-CoA and is uncompetitive versus NADPH, and that NADP+ is competitive versus NADPH and uncompetitive versus acetyl-CoA or malonyl-CoA. These results are consistent with a seven-site ping-pong mechanism with intermediates covalently bound to 4'-phosphopantetheine (part of acyl carrier protein). Double competitive substrate inhibition by acetyl-CoA and malonyl-CoA is consistent with the rate equation derived for the over-all mechanism. The kinetic mechanism developed from these results is capable of explaining the formation of fatty acids from malonyl-CoA and NADPH alone (Katiyar, S. S., Briedis, A. V., and Porter, J. W. (1974) Arch. Biochem. Biophys. 162, 412-420) and also the formation of triacetic acid lactone from either malonyl-CoA alone or acetyl-CoA plus malonyl-CoA.     

Graphic rules in steady and non-steady state enzyme kinetics

Graphic methods, when applied to enzyme kinetics, can provide a visually intuitive relation between calculations and reaction graphs. This will not only greatly raise the efficiency of calculations but also significantly help the analysis of enzyme kinetic mechanisms. In this paper, four graphic rules are presented. Rules 1-3 are established for steady state enzyme-catalyzed reaction systems and Rule 4 is for non-steady state ones. In comparison with conventional graphic methods which can only be applied to steady state systems, the present rules have the following merits. 1) Complicated and tedious calculations can be greatly simplified; for example, in calculating the concentrations of enzyme species for the bi-bi random mechanism, the calculation work can be reduced 8-fold compared with the King-Altman's method. 2) A great deal of wasted labor can be avoided; for example, in calculating the rate of product formation for the same mechanism, the operation of finding and removing the 96 reciprocally canceled terms is no longer needed because they automatically disappear during the derivation. 3) Final results can be easily and safely checked by a formula provided in each of the graphic rules. 4) Non-steady state systems can also be treated by the present graphic method; for example, applying Rule 4, one can directly write out the solution for a non-steady state enzyme-catalyzed system, without the need to follow more difficult and complicated operations to solve differential equations. The mathematical proofs of Rules 1-4 are given in Appendices A-D (in the Miniprint), respectively.     

Integrated stoichiometric, thermodynamic and kinetic modelling of steady state metabolism

The quantitative analysis of biochemical reactions and metabolites is at frontier of biological sciences. The recent availability of high-throughput technology data sets in biology has paved the way for new modelling approaches at various levels of complexity including the metabolome of a cell or an organism. Understanding the metabolism of a single cell and multi-cell organism will provide the knowledge for the rational design of growth conditions to produce commercially valuable reagents in biotechnology. Here, we demonstrate how equations representing steady state mass conservation, energy conservation, the second law of thermodynamics, and reversible enzyme kinetics can be formulated as a single system of linear equalities and inequalities, in addition to linear equalities on exponential variables. Even though the feasible set is non-convex, the reformulation is exact and amenable to large-scale numerical analysis, a prerequisite for computationally feasible genome scale modelling. Integrating flux, concentration and kinetic variables in a unified constraint-based formulation is aimed at increasing the quantitative predictive capacity of flux balance analysis. Incorporation of experimental and theoretical bounds on thermodynamic and kinetic variables ensures that the predicted steady state fluxes are both thermodynamically and biochemically feasible. The resulting in silico predictions are tested against fluxomic data for central metabolism in Escherichia coli and compare favourably with in silico prediction by flux balance analysis.     

A steady state model of sequential irreversible enzyme reactions

Summary One of the questions which arises in the study of certain inborn errors of metabolism as well as in the field of enzyme kinetics is: what are the quantitative relationships between parameters of enzyme activity and substrate pool sizes in a metabolic pathway? A steady state model has been devised to answer this question for a homogeneous system of non-branched sequential irreversible enzyme reactions which follow Michaelis-Menten kinetics. The concentration of a substrate in such a pathway, [S_i], is a function of 5 variables: (a) the K_M of the enzyme which forms the substrate (K_{M (i–1)}), (b) the K_M of the enzyme which utilizes the substrate (K_{M i}), (c) the V_max of the enzyme which forms the substrate (V_{m (i–1)}), (d) the V_max of the enzyme which utilizes the substrate (V_{m i}) and (e) the immediate precursor concentration [S_(i–1)] where [S_i] = K_{M i} V_{m (i–1)} [S_(i–1)]/[S_(i–1)] (V_mi -V_{m (i–1)}) + K_{M (i–1)}) V_mi The model introduces and defines the concept of and conditions for amplification. An input in the form of a steady state concentration of precursor [S_(i–1)] may be amplified as an output in the form of an increased steady state concentration of product [S_i]. The model also defines the values of the above 5 parameters which do not allow attainment of a steady state for the type of pathway considered.From the Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014.     

Statistical determination of steady state condition in bioremediation tests

Linear regression and two newly developed statistical techniques were used to determine steady states in the dependent y-variables (effluent concentration or removal rate of pollutants) using correlation coefficients (r) for the relationship between the independent x-variables (reactor operating or treatment time) and the dependent y-variables. The statistical technique applied to chlorophenol bioremediation using a varying number of data points for linear regression analysis was more useful in determining a steady state for six general data patterns from bioremediation tests than the statistical technique using a fixed number of data points for linear regression analysis.     

Determination of kinase activity by using an enzyme electrode

A method for determining the enzymatic activity of hexokinases, acetate kinase and pyruvate kinase using an enzyme electrode was developed. The assay time is 2-3 min. The lower limit of the activity determining is 0,054 U/ml. The proposed method was applied to investigation of pyruvate kinase and acetate kinase reactivation under the action of mercaptoethanol.     

Design and response characteristics of an enzyme electrode for measurement of penicillin in fermentation broth

A principle for the construction of an autoclavable enzyme electrode is presented. Some characteristics of a penicillin electrode constructed according to this principle are given. The response time is ˜1 min. The response to increasing concentrations of penicillin is linear in buffered samples but logarithmic in unbuffered samples. The reason for the linearity is discussed. A local pH decrease in the enzyme, which is a is a consequence of the enzymatic reaction, reduces the buffering capacity within the electrode and thus increases the sensitivity. It is suggested that this increased sensitivity eliminates the logarithmic response predicted by the Nernst equation for a pH-based enzyme electrode.     

Purification and steady state kinetic mechanism of glycogen synthase-D from human polymorpho-nuclear leukocytes

Summary The authors' work on the purification and steady state kinetic investigation of the enzyme glycogen synthase D (UDP-glucose: glycogen 4--glucosyl-transferase, EC 2.4.1.11) from human polymorphonuclear leukocytes is reviewed. The main features of the kinetic mechanism for catalysis of the reaction UDPG + glycogen_n UDP + glycogen_(n+1) are: (i) Lineweaver-Burk plots in both substrates are linear, exhibiting intersecting patterns; (ii) UDP is a competitive, respectively noncompetitive, inhibitor towards the substrates UDPG and glycogen; (iii) the essential activator glucose-6-phosphate (G-6-P) showed an intersecting pattern towards glycogen and an equilibrium ordered pattern towards UDPG. These features identify in this case the mechanism as a rapid equilibrium random bi-bi mechanism, with G-6-P adding to the enzyme prior to the substrate UDPG. New results on the influence of the modifiers NaCl, Ca⁺⁺, Mn⁺⁺, Mg⁺⁺, HPO₄ ^–-, SO₄ ^–-, and ATP on the enzyme are reported. Interpreting the observations in terms of the established mechanism, the following results are obtained: The effect of salt (NaCl) is nonspecific and fairly small, probably reflecting a general action of the electrolyte medium on the conformation of the enzyme. Divalent cations affect only the rate limiting step, i.e. the interconversion of the quaternary enzyme-substrate-activator complexes. The anions interact exclusively with the G-6-P binding site of the enzyme. The dissociation constants for the enzyme-modifier complexes are determined, and a kinetic mechanism for the action of the anions is proposed, leading to activation or inhibition, depending on the concentration of G-6-P.An invited article     

A possible in vivo mechanism of intermediate transfer by glycolytic enzyme complexes: steady state fluorescence anisotropy analysis of an enzyme complex formation.

Rate constants of dissociation (k(off)) and association (k(on)) of the bienzyme complex yeast glyceraldehyde-3-phosphate dehydrogenase--yeast alcohol dehydrogenase have been determined in the absence and presence of NAD or NADH by fluorescence anisotropy measurements. We found that dissociation of the complex is considerably slower than catalytic turnover of either of the enzymes (that is k(off) much less than kcat) irrespective of the presence of coenzymes. A perusal of the literature reveals that this relation invariably applies to all systems studied so far. These observations all taken together constitute compelling evidence that direct metabolite transfer in enzyme complexes cannot be satisfactorily described by invoking the dynamic model but requires a model assuming more lasting complexes. This seems to support the case of the temporary-stationary model suggested by one of us. Implications of this conclusion are treated in depth and further evidence is cited under Discussion.