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.     

Half-time analysis of the kinetics of irreversible enzyme inhibition by an unstable site-specific reagent

The half-time method for the determination of Michaelis parameters from enzyme progress-curve data (Wharton, C.W. and Szawelski, R.J. (1982) Biochem. J. 203, 351-360) has been adapted for analysis of the kinetics of irreversible enzyme inhibition by an unstable site-specific inhibitor. The method is applicable to a model in which a product (R) of the decomposition of the site-specific reagent, retaining the chemical moiety responsible for inhibitor specificity, binds reversibly to the enzyme with dissociation constant Kr: (formula; see text). Half-time plots of simulated enzyme inactivation time-course data are shown to be unbiased, and excellent estimates of the apparent second-order rate constant for inactivation (k +2/Ki) and Kr can be obtained from a series of experiments with varying initial concentrations of inhibitor. Reliable estimates of k +2 and Ki individually are dependent upon the relative magnitudes of the kinetic parameters describing inactivation. The special case, Kr = Ki, is considered in some detail, and the integrated rate equation describing enzyme inactivation shown to be analogous to that for a simple bimolecular reaction between enzyme and an unstable irreversible inhibitor without the formation of a reversible enzyme-inhibitor complex. The half-time method can be directly extended to the kinetics of enzyme inactivation by an unstable mechanism-based (suicide) inhibitor, provided that the inhibitor is not also a substrate for the enzyme.     

Kinetics of suicide substrates. Practical procedures for determining parameters.

Many clinically important or mechanistically interesting inhibitors react with enzymes by a branched pathway in which inactivation of the enzyme and formation of product are competing reactions. The steady-state kinetics for this pathway [Waley (1980) Biochem. J. 185, 771-773] gave equations for progress curves that were cumbersome. A convenient linear plot is now described. The time (t1/2) for 50% inactivation of the enzyme (this is also the time for 50% formation of product), or for 50% loss of substrate, is measured in a series of experiments in which the concentration of inhibitor, [I]0, is varied; in these experiments the ratio of the concentration of enzyme to the concentration of inhibitor is kept fixed. Then a plot of [I]0 X t1/2 against [I]0 is linear, and the kinetic parameters can be found from the slope and intercept. Furthermore, simplifications of the equations for progress curves are described that are valid when the concentration of inhibitors is high, or is low, or when the extent of reaction is low. The use of simulated data has shown that the recommended methods are not unduly sensitive to experimental error.     

Progress curve analysis of adenosine deaminase-catalyzed reactions

The kinetic constants of the adenosine deaminase-catalyzed conversion of adenosine to inosine were found to be readily obtainable by analyzing the progress curve of a single reaction. A novel inhibitor, 9-(1-hydroxymethyl-3-methylbutyl)adenine, was studied to test the validity of the progress curve method with this enzyme. Estimates of kinetic constants determined by this method were compared to those determined by the conventional initial velocity analysis. The Km and Vmax values for adenosine and the Ki value for the inhibitor were estimated to be 26.1 microM, 1.27 mumol/min/unit of enzyme, and 0.48 microM, respectively, by the initial velocity method, and 29.3 microM, 1.27 mumol/min/unit of enzyme, and 0.52 microM, respectively, by the progress curve analysis. The inhibitor was shown to act competitively with substrate by both methods of analysis. The progress curve experiments were very simple to perform and the constants were calculated (with an interfaced microcomputer) within a few minutes of the completion of each assay.     

Transient-phase studies on the creatine kinase reaction. The analysis of a reaction pathway with three intermediates.

1. The initial formation of creatine phosphate by creatine kinase was studied in the millisecond range and the effect of temperature on the transient and steady-state phases exploited. 2. At 25 degrees C and 35 degrees C there was no transient phase. This is in agreement with the results of Gutfreund [Engelborghs, Y., Marsh, A., and Gutfreund, H. (1975) Biochem. J. 151, 47--50]. 3. At 4 degrees C the time course of creatine phosphate formation was complex and consisted of three transient phases: a lag phase, a burst phase and a steady-state phase. Based on this result a reaction scheme for creatine kinase which includes three intermediates was proposed. Despite the completeness of the time course, the extraction of estimates for the rate constants was difficult and computer simulation and iterative methods had to be resorted to. 4. Attempts were made to provide evidence for the complex enzyme.ADP.metaphosphate.creatine on the creatine kinase reaction pathway [cf. Milner-White, E.J. and Watts, D.C. (1971) Biochem. J. 122, 727--740]. Under the conditions used these attempts were unsuccessful at times down to 2.5 ms, at 4 degrees C or 35 degrees C.     

Enzyme kinetic studies from progress curves.

A method is described for fitting the velocities obtained from progress curves to a steady-state rate equation. It is based on the method of Markus & Plesser [(1981) in Kinetic Data Analysis: Design and Analysis of Enzyme and Kinetic Data (Edrenyi, ed.), pp. 317-339, Plenum Press, New York]. The obstacle of needing good initial estimates of kinetic parameters is removed by using the parameters provided graphically by a minor modification of the method of Yun & Suelter [(1977) Biochim, Biophys. Acta 480, 1-13]. This progress-curved-based method allows the same discrimination among rival models as do the initial-velocity-based methods, with a great saving of experimental time. The BASIC and FORTRAN 77 programs are deposited as Supplementary Publication SUP 50132 (17 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5-6.     

Kinetic studies on the reaction catalysed by phosphofructokinase from Trypanosoma brucei.

The steady-state kinetics of the reaction catalysed by the bloodstream form of Trypanosoma brucei were studied at pH 6.7. In the presence of 50 mM-potassium phosphate buffer, the apparent co-operativity with respect to fructose 6-phosphate and the non-linear relationship between initial velocity and enzyme concentration, which were found when the enzyme was assayed in 50 mM-imidazole buffer [Cronin & Tipton (1985) Biochem. J. 227, 113-124], are not evident. Studies on the variations of the initial rate with changing concentrations of MgATP and fructose 6-phosphate, the product inhibition by fructose 1,6-bisphosphate and the effects of the alternative substrate ITP were consistent with an ordered reaction pathway, in which MgATP binds to the enzyme before fructose 6-phosphate, and fructose 1,6-bisphosphate is the first product to dissociate from the ternary complex.     

The mechanism of action of xanthine oxidase. The relationship between the rapid and very rapid molybdenum electron-paramagnetic-resonance signals.

On the basis of the work of Gutteridge, Tanner & Bray [Biochem. J. (1978) 175, 887-897] and of other data in the literature, a mechanism for the reaction of xanthine oxidase with reducing substrates is proposed. In the Michaelis complex, xanthine is bound to molybdenum via the N-9 nitrogen atom. Coupled transfer of two electrons to molybdenum and the C-8 proton to the enzyme yields (Enzyme)-Mo-SH. Concerted with this process, reaction of the xanthine residue with a nucleophile in the active centre yields a covalent intermediate that breaks down to give the product by alternative pathways at high and at low pH values.     

Substrate depletion analysis as an approach to the pre-steady-state anticooperative kinetics of aminoacyl adenylate formation by tryptophanyl-tRNA synthetase from beef pancreas

The formation of tryptophanyl adenylate catalyzed by tryptophanyl-tRNA synthetase from beef pancreas has been studied by stopped-flow analysis under conditions where the concentration of one of the substrates was largely decreasing during the time course of the reaction. Under such conditions a nonlinear regression analysis of the formation of the adenylate (adenylate vs. time curve) at several initial tryptophan and enzyme concentrations gave an accurate determination of both binding constants of this substrate. The use of the jackknife procedure according to Cornish - Bowden & Wong [ Cornish - Bowden , A., & Wong , J.J. (1978) Biochem. J. 175, 969-976] gave the limit of confidence of these constants. This approach confirmed that tryptophanyl-tRNA synthetase presents a kinetic anticooperativity toward tryptophan in the activation reaction that closely parallels the anticooperativity found for tryptophan binding at equilibrium. Both sites are simultaneously forming the adenylate. The dissociation constants obtained under the present pre-steady-state conditions for tryptophan are KT1 = 1.6 +/- 0.5 microM and KT2 = 18.5 +/- 3.0 microM at pH 8.0, 25 degrees C. The rate constant kf of adenylate formation is identical for both active sites (kf = 42 +/- 5 s-1). The substrate depletion method presently used, linked to the jackknife procedure, proves to be particularly suitable for the determination of the kinetic constants and for the discrimination between different possible kinetic models of dimeric enzyme with high substrate affinity. In such a case this method is more reliable than the conventional method using substrate concentrations in high excess over that of the enzyme.     

Reconstitution of monomeric cytochrome c oxidase into phospholipid vesicles yields functionally interacting cytochrome aa3 units

When the carbon monoxide complex of fully reduced cytochrome c oxidase, reconstituted into liposomes, is mixed with oxygen-containing buffer, complex kinetic progress curves are observed. This pattern is seen irrespective of whether the oxidase used in reconstitution is the dimeric or monomeric (subunit III-depleted) enzyme. These findings are interpreted in the light of similar experiments on the detergent-solubilized enzyme reported by Gibson and Greenwood (Gibson, Q.H., and Greenwood, C. (1963) Biochem. J. 86, 541-554) and confirmed by ourselves. We conclude that reconstitution of monomeric (subunit III-less) enzyme yields, preferentially, vesicles containing more than one functional unit, possibly associated as dimers. This result is of significance to our understanding of the relationships between aggregation state and proton pumping capacity of cytochrome oxidase.     

Application of 19F nuclear magnetic resonance to examine covalent modification reactions of tyrosyl derivatives: a study of calcineurin catalysis

The hydrolysis of fluorotyrosine phosphate by the calmodulin-activated phosphatase calcineurin has been monitored by 19F nuclear magnetic resonance spectroscopy. Previous work had established that the 19F nuclear magnetic resonance shift of the fluorine nucleus was altered after the phosphorylation of the phenolic hydroxyl group (B. Martin, C.J. Pallen, J.H. Wang, and D.J. Graves (1985) J. Biol. Chem. 260, 14592-14597). The disappearance of substrate and the appearance of product can be measured simultaneously with this approach. Application of the integrated form of the Michaelis-Menten equation yields estimates of the kinetic parameter, KM, close to the values obtained by initial rate kinetics. The velocity term, VM, was also evaluated to be approximately the same value. Calcineurin was determined not to be inactivated over the time period of the reaction. The results demonstrate that 19F nuclear magnetic resonance spectroscopy can be applied to the examination of enzyme-catalyzed reactions.     

Analysis of progress curves. Interaction of pyruvate kinase from Escherichia coli with fructose 1,6-bisphosphate and calcium ions.

The influence of fructose 1,6-bisphosphate and Ca2+ on the kinetics of pyruvate kinase from Escherichia coli K12 was studied (at pH 7.0 and 25 degrees C) by using the pH-stat method for the measurement of the reaction progress as well as initial-rate analysis. The data were analysed on the basis of a concerted model with three conformational states [Markus, Plesser, Boiteux, Hess & Malcovati (1980) Biochem. J. 189, 421-433] by using a novel procedure for a computer-directed treatment of progress curves [Markus & Plesser (1976) Biochem. Soc. Trans. 4, 361-364]. By addition of fructose 1,6-bisphosphate the sigmoid kinetics with respect to phosphoenolpyruvate and Mg2+ is abolished and the activity of the enzyme is described by classical saturation kinetics. This is explained by exclusive binding of fructose 1,6-bisphosphate at an allosteric site of the conformational state that forms the active complex. We observe that Ca2+ is an activator of the enzyme at low Mg2+ and Ca2+ concentrations; otherwise it is an inhibitor. These effects can be understood by assuming that Ca2+ has the same binding properties as Mg2+, although it does not allow a catalytic turnover.     

An automated method to evaluate the enzyme kinetics of β‐glucosidases

Enzyme kinetic measurements are important for the characterization and engineering of biocatalysts, with applications in a wide range of research fields. The measurement of initial reaction velocity is usually slow and laborious, which motivated us to explore the possibilities for automating this process. Our model enzyme is the maize β‐glucosidase Zm‐p60.1. Zm‐p60.1 plays a significant role in plant growth and development by regulating levels of the active plant hormone cytokinin. Zm‐p60.1 belongs to a wide group of hydrolytic enzymes. Members of this group hydrolyze several different types of glucosides, releasing glucose as a secondary product. Enzyme kinetic measurements using artificial substrates are well established, but burdensome and time‐consuming. Thus, they are a suitable target for process automation. Simple optical methods for enzyme kinetic measurements using natural substrates are often impossible given the optical properties of the enzymatic reaction products. However, we have developed an automated method based on glucose detection, as glucose is released from all substrates of glucosidase reactions. The presented method can obtain 24 data points from up to 15 substrate concentrations to precisely describe the enzyme kinetics. The combination of an automated liquid handling process with assays that have been optimized for measuring the initial hydrolysis velocity of β‐glucosidases yields two distinct methods that are faster, cheaper, and more accurate than the established protocols.     

Carotenoid Chemistry and Biochemistry : Edited by G. Britton and T.W. Goodwin Pergamon Press; Oxford, 1982 ix + 399 pages. £47.50

The dimeric tryptophanyl-tRNA synthetase from beef pancreas has been found to activate 2 tryptophans/mol enzyme [Eur. J. Biochem. (1982) 128, 389–398]. By using quenched-flow and stopped-flow methods under presteady-state conditions, we show that only one enzyme subunit operates at a time in the aminoacylation of tRNA^Trp and that the transfer reaction is not the rate-limiting step in the overall aminoacylation process.     

About the pKa of the active-site histidine in flavocytochrome b2 (yeast L-lactate dehydrogenase).

Flavocytochrome b2 or L-lactate dehydrogenase from yeasts catalyzes the oxidation of L-lactate at the expense of monoelectronic acceptors such as cytochrome c, its physiological partner. When incubated in the presence of both L-lactate and a keto acid, the enzyme catalyzes a transhydrogenation reaction wherein only the flavin is involved. During this reaction, the substrate alpha-hydrogen is transferred not only to the solvent but also in part to the keto acid, which acts as reverse substrate. Thus, when bound to the reduced enzyme, this hydrogen is sticky. In the context of a carbanion mechanism, it resides on Nepsilon of His373, the active site base. We have shown before that a correlation between the amount of intermolecular hydrogen transfer from [2-3H] lactate and the keto acid reverse substrate concentration enables the determination of the first-order rate constant, kHe, for exchange of the substrate-derived protein-bound hydrogen with bulk solvent (Urban P, Lederer F, 1985, J Biol Chem 260:11115-11122). In this work, we show that the exchange with the solvent appears to be independent of the phosphate buffer concentration in the range from 40 to 500 mM. It is thus probable that exchange occurs directly with water molecules. The second-order rate constant for exchange is then 0.16 (+/-0.03) M(-1) s(-1). Using the Eigen equation, this figure yields a pKa of 9.1+/-0.1 for His373 in the reduced enzyme, compared to a probable value of 6.0 or less in the oxidized enzyme (Suzuki H, Ogura YC, 1970, J Biochem 67:291-295). The mechanistic significance of these results is discussed.     

A computer program for enzyme kinetics that combines model discrimination, parameter refinement and sequential experimental design.

A method of model discrimination and parameter estimation in enzyme kinetics is proposed. The experimental design and analysis of the model are carried out simultaneously and the stopping rule for experimentation is deduced by the experimenter when the probabilities a posteriori indicate that one model is clearly superior to the rest. A FORTRAN77 program specifically developed for joint designs is given. The method is very powerful, as indicated by its usefulness in the discrimination between models. For example, it has been successfully applied to three cases of enzyme kinetics (a single-substrate Michaelian reaction with product inhibition, a single-substrate complex reaction and a two-substrate reaction). By using this method the most probable model and the estimates of the parameters can be obtained in one experimental session. The FORTRAN77 program is deposited as Supplementary Publication SUP 50134 (19 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5.     

Glucose-6-phosphate dehydrogenase from Dicentrarchus labrax liver: kinetic mechanism and kinetics of NADPH inhibition

The kinetic mechanism of the reaction catalyzed by glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from Dicentrarchus labrax liver was examined using initial velocity studies, NADPH and glucosamine 6-phosphate inhibition and alternate coenzyme experiments. The results are consistent with a steady-state ordered sequential mechanism in which NADP+ binds first to the enzyme and NADPH is released last. Replots of NADPH inhibition show an uncommon parabolic pattern for this enzyme that has not been previously described. A kinetic model is proposed in agreement with our kinetic results and with previously published structural studies (Bautista et al. (1988) Biochem. Soc. Trans. 16, 903-904). The kinetic mechanism presented provides a possible explanation for the regulation of the enzyme by the [NADPH]/[NADP+] ratio.     

The affinity of rabbit muscle fructose-bisphosphatase for fructose bisphosphate

This paper reports that microM concentrations of fructose bisphosphate are titrated by rabbit muscle fructose-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) when the enzyme concentration is varied in the range which secures measurable initial velocities of reaction: a result that can only be explained by supposing that the enzyme has a greater affinity for fructose bisphosphate than suggested by Fernando, J., Enser, M., Pontremoli, S. and Horecker, B.L. (1968) Arch. Biochem. Biophys. 126, 599-606. The results also suggest that the keto form of the substrate may be the preferred configuration and that the enzyme is inhibited by magnesium-bound fructose bisphosphate.     

The nature of the allosteric interactions of ribonuclease and its ligands.

The allosteric model for ribonuclease activity by Walker, Ralston & Darvey [(1975) Biochem.J. 147, 425--433; (1976) Biochem.J. 153, 329--337] involves the binding of a large number of molecules of substrate or substrate analogue to a series of allosteric sites on the enzyme. In the present paper, the nature of these allosteric interactions is investigated. The effects of ionic strength pH carbamoylation of lysine to homocitrulline and of deamidation of glutamine and asparagine on plots of velocity versus substrate concentration are examined and evidence is presented that the allosteric transition involves an electrostatic interaction between the negatively charged substrate molecules and the cationic groups on the enzyme.     

The analysis of enzyme progress curves by numerical differentiation, including competitive product inhibition and enzyme reactivation

A new method for analyzing steady-state enzyme kinetic data is presented. The technique, which is based on the numerical differentiation of the complete reaction curve, has several advantages over initial velocity and integrated Michaelis-Menten equation methods. The differentiated data are fit to the differential equation describing the appropriate kinetic scheme. This approach is particularly valuable in cases of strong competitive product inhibition and of changing concentrations of active enzyme. The method assumes a reversible reaction and is applicable to a very wide variety of steady-state kinetic schemes. A particular advantage of this approach over integrated methods is that it is independent of [S0] and hence of errors in [S0]. The combination of complete progress curve and computer analysis makes this approach very efficient with respect to both time and materials. Running on an IBM PC XT or equivalent microcomputer with an 8087 coprocessor, the analyses are very fast, the complete process usually being complete in a minute or two. The utility of the technique is demonstrated by application to both simulated and real data. We show that the differentiation of the progress curve for the ribonuclease-catalyzed hydrolysis of 2',3'-cyclic cytidine monophosphate reveals strong product inhibition by 3'-CMP, and this product inhibition accounts for the large discrepancies reported in the literature for the value of Km for this substrate. The method was also applied to determine the rate of reactivation of beta-lactamase which had been reversibly inactivated by cloxacillin. Since large numbers of data points are required for the numerical differentiation the method has become practical only with the advent of computer-acquired data systems.