Chemical mechanism of lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung. pH-dependence of kinetic parameters.

Lysophosphatidylcholine: lysophosphatidylcholine acyltransferase is an enzyme that catalyses two reactions: hydrolysis of lysophosphatidylcholine and transacylation between two molecules of lysophosphatidylcholine to give disaturated phosphatidylcholine. Following the kinetic model previously proposed for this enzyme [Martín, Pérez-Gil, Acebal & Arche (1990) Biochem. J. 266, 47-53], the values of essential pK values in free enzyme and substrate-enzyme complexes have now been determined. The chemical mechanism of catalysis was dependent on the deprotonation of a histidine residue with pK about 5.7. This result was supported by the perturbation of pK values by addition of organic solvent. Very high and exothermic enthalpy of ionization was measured, indicating that a conformational re-arrangement in the enzyme accompanies the ionization of the essential histidine residue. These results, as well as the results from previous studies, enabled the proposal of a chemical mechanism for the enzymic reactions catalysed by lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung.     

Lysolecithin:lysolecithin acyltransferase from rabbit lung. A conformational study

The enzyme lysolecithin:lysolecithin acyltransferase from rabbit lung has been found to have a relatively disordered conformation in solutions of high ionic strength. The protein exhibited an ordering of structure when salt was suppressed. This conformational change was concomitant with the loss of transacylase activity, the hydrolytic reaction remaining unchanged. Addition of NaCl caused a progressive disordering of structure with a parallel increase of transacylase activity. The acid denaturation of the protein, at low and high ionic strengths, showed that the ionization of groups with pK in the range 5.9-6.4 was essential for denaturation. The structure was stable at basic pH. The addition of lipids resulted in a non-specific stabilization of the disordered conformation, in the same manner as the addition of NaCl. From these results, it is suggested that there are two conformations for this protein which differ in their ability to bind lysolecithin molecules in the enzyme deacylation step of the reaction. This hypothesis agrees with previously published properties of the enzyme, concerning aggregation with other proteins and kinetic data. From the amino acid composition and conformational properties, the authors suggest that this enzyme could be a peripheral membrane protein.     

d-3-Hydroxybutyrate dehydrogenase from Rhodopseudomonas spheroides. Kinetic mechanism from steady-state kinetics of the reaction catalysed by the enzyme in solution and covalently attached to diethylaminoethylcellulose

1. The reversible NAD(+)-linked oxidation of d-3-hydroxybutyrate to acetoacetate in 0.1m-sodium pyrophosphate buffer, pH8.5, at 25.0 degrees C, catalysed by d-3-hydroxybutyrate dehydrogenase (d-3-hydroxybutyrate-NAD(+) oxidoreductase, EC 1.1.1.30), was studied by initial-velocity, dead-end inhibition and product-inhibition analysis. 2. The reactions were carried out on (a) the soluble enzyme from Rhodopseudomonas spheroides and (b) an insoluble derivative of this enzyme prepared by its covalent attachment to DEAE-cellulose by using 2-amino-4,6-dichloro-s-triazine as coupling agent. 3. The insolubilized enzyme preparation contained 5mg of protein/g wet wt. of total material, and when freshly prepared its specific activity was 1.2mumol/min per mg of protein, which is 67% of that of the soluble dialysed enzyme. 4. The reactions catalysed by both the enzyme in solution and the insolubilized enzyme were shown to follow sequential pathways in which the nicotinamide nucleotides bind obligatorily first to the enzyme. Evidence is presented for kinetically significant ternary complexes and that the rate-limiting step(s) of both catalyses probably involves isomerization of the enzyme-nicotinamide nucleotide complexes and/or dissociation of the nicotinamide nucleotides from the enzyme. Both catalyses therefore are probably best described as ordered Bi Bi mechanisms, possibly with multiple enzyme-nicotinamide nucleotide complexes. 5. The kinetic parameters and the calculable rate constants for the catalysis by the soluble enzyme are similar to the corresponding parameters and rate constants for the catalysis by the insolubilized enzyme.     

Mechanism of the reaction catalyzed by acyl-CoA: lysolecithin acyltransferase from rabbit lung. pH studies and chemical modification

This paper deals with the first attempt to elucidate the chemical mechanism of acyl-CoA: lysolecithin acyltransferase from rabbit lung, a key enzyme in the metabolism of lung surfactant. For this purpose, the pH dependence of kinetic constants as well as the chemical modification of the protein have been studied on a partially-purified preparation. From these experiments, the pKs on which the activity of the enzyme relies have been calculated, giving values of pK1 congruent to 5.5 and pK2 congruent to 10. Analysis of the effect of organic solvents on these pKs and the calculation of the enthalpies of ionization, together with the chemical modification experiments, lead to the conclusion that pK1 is due to an histidine residue, whereas pK2 arises from the amino group of the adenine ring of palmitoyl-CoA. Moreover, chemical modification demonstrated an essential cysteine. A tentative chemical mechanism, in accordance with these results, is proposed and it is hypothesized, in view of other results obtained in our laboratory and from the literature, that the chemical mechanism of acyl transfer to sn-2 position may be common to other enzymes of glycerolipid metabolism.     

A 'branched' mechanism of the reverse reaction of yeast glutathione reductase. An estimation of the enzyme standard potential values from the steady-state kinetics data

The reduced glutathione-linked NADP+ reduction, catalyzed by yeast glutathione reductase, follows a 'sequential' or 'ping-pong' mechanism at high or low NADP+ concentrations, respectively. The pattern of the NADPH and NADP+ cross-inhibition reflects not only the competition for the binding site, but the shift of the reaction equilibrium as well. A 'branched' scheme of the glutathione reductase reaction is presented. The enzyme standard potential (-255 mV, pH 7.0) was estimated from the ratio of the NADPH and NADP+ rate constants corresponding to the ping-pong mechanism.     

Kinetic mechanism from steady-state kinetics of the reaction catalysed by baker''s-yeast glucose 6-phosphate dehydrogenase in solution and covalently attached to sepharose.

1. The reaction catalysed by glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast was studied in 42mM-glycylglycine buffer, pH7.4 at 25 degrees C, by initial-velocity studies and by the use of NADPH as a product inhibitor. 2. The reactions catalysed by both the soluble enzyme and a stable enzyme covalently attached to CNBr-activated Sepharose 4B probably follow an ordered reaction mechanism with NADP+ and NADPH as the leading reactants. 3. The kinetic constants obtained for the soluble enzyme lere: KNADP+m, 19 muM; KNADP+s, 23 muM; KNADPHs, 15 muM. Similar values were obtained for the immobilized enzyme. 4. The assay of the immobilized enzyme was done by using a micro packed-bed recirculation reactor, and the advantages of this technique are discussed.     

Analysis of the mechanism of chloramphenicol acetyltransferase by steady-state kinetics. Evidence for a ternary-complex mechanism.

The mechanism of the enzymic reaction responsible for chloramphenicol resistance in bacteria was examined by steady-state kinetic methods. The forward reaction catalysed by chloramphenicol acetyltransferase leads to inactivation of the antibiotic. Use of alternative acyl donors and acceptors, as well as the natural substrates, has yielded data that favour the view that the reaction proceeds to the formation of a ternary complex by a rapid-equilibrium mechanism wherein the addition of substrates may be random but a preference for acetyl-CoA as the leading substrate can be detected. Chloramphenicol and acetyl-CoA bind independently, but the correlation between directly determined and kinetically derived dissociation constants is imperfect because of an unreliable slope term in the rate equation. The reverse reaction, yielding acetyl-CoA and chloramphenicol, was studied in a coupled assay involving citrate synthase and malate dehydrogenase, and is best described by a rapid-equilibrium mechanism with random addition of substrates. The directly determined dissociation constant for CoA is in agreement with that derived from kinetic measurements under the assumption of an independent-sites model.     

Imidazole glycerol phosphate synthase from Thermotoga maritima. Quaternary structure, steady-state kinetics, and reaction mechanism of the bienzyme complex

Imidazole glycerol phosphate synthase, which links histidine and de novo purine biosynthesis, is a member of the glutamine amidotransferase family. In bacteria, imidazole glycerol phosphate synthase constitutes a bienzyme complex of the glutaminase subunit HisH and the synthase subunit HisF. Nascent ammonia produced by HisH reacts at the active site of HisF with N'-((5'-phosphoribulosyl)formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide to yield the products imidazole glycerol phosphate and 5-aminoimidazole-4-carboxamide ribotide. In order to elucidate the interactions between HisH and HisF and the catalytic mechanism of the HisF reaction, the enzymes tHisH and tHisF from Thermotoga maritima were produced in Escherichia coli, purified, and characterized. Isolated tHisH showed no detectable glutaminase activity but was stimulated by complex formation with tHisF to which either the product imidazole glycerol phosphate or a substrate analogue were bound. Eight conserved amino acids at the putative active site of tHisF were exchanged by site-directed mutagenesis, and the purified variants were investigated by steady-state kinetics. Aspartate 11 appeared to be essential for the synthase activity both in vitro and in vivo, and aspartate 130 could be partially replaced only by glutamate. The carboxylate groups of these residues could provide general acid/base catalysis in the proposed catalytic mechanism of the synthase reaction.     

Evidence of a pH-dependent conformational change at the active site of lysolecithin:lysolecithin acyltransferase from rabbit lung

It has been shown that both activities, hydrolysis and transacylation, of lysolecithin:lysolecithin acyltransferase, as well as the conformation of the polypeptide are critically dependent on a pK around 5.8, but the question remains if the same residue(s) is responsible for the conformational change and the loss of activity. In this paper, ultrasonic cavitation is used to study the pH-dependent inactivation. The results show that there are two first-order inactivation constants which depend on pH and that the transition between them has a pK of 5.9. As the constants of ultrasonic inactivation are very dependent on the accessibility of the residues it is concluded that the conformational change modifies the accessibility of the active site.     

Statistical analyses of enzyme inhibitor kinetics: a hierarchical model-dependent approach.

Inference of the nature of an enzyme inhibitor and calculations of inhibitor constants from linear plots are prone to be unreliable when experimental error is significant. This may be minimised by a method of statistical analysis using nonlinear regression methods and modelling using the variance ratio test of a hierarchy of models of inhibitor behaviour.     

High-pressure enzyme kinetics. Lactate dehydrogenase in an optical cell that allows a reaction to be started under high pressure

Interactions of adriamycin with ferritin-bound iron have been investigated. It is demonstrated (i) that adriamycin stimulates an iron-dependent lipid peroxidation in submitochondrial particles in the presence of ferritin, and (ii) that incubation of adriamycin with ferritin results in a slow transfer of iron to adriamycin with formation of an adriamycin-iron complex. The results are discussed in relation to the possible role for intracellular iron in adriamycin toxicity.     

The accuracy of the rapid equilibrium assumption in steady-state enzyme kinetics in the case of a multipath arbitrary enzyme mechanism with a number of equilibrium segments

A quantitative evaluation of the accuracy of the rapid-equilibrium assumption in steady-state enzyme kinetics was obtained for a multipath arbitrary enzyme mechanism with a number of equilibrium segments. Explicit expressions for estimating the contribution of any equilibrium segment to the accuracy of the rapid-equilibrium assumption were obtained. This allowed us to determine the accuracy of the rapid-equilibrium assumption (Δ) in general: 1 + Δ = (1 + Δ₁)(1 + Δ₂)... (1 + Δ_k), where Δ₁, Δ₂,..., Δ_k is the contribution of each individual equilibrium segment. The accuracy depends only on the structure and properties of equilibrium segments, which have been accounted for in the rapid-equilibrium assumption, but it is independent of the number of paths in the mechanism of the enzymatic reaction and on the structure and properties of the remaining part (steady-state) of the kinetic scheme.     

On the application of the Clark oxygen electrode to the study of enzyme kinetics in apolar solvents: the catalase reaction.

A method for recording O2 concentrations in nonconducting organic media with the Clark oxygen electrode was developed. Spontaneous oxidation of Na2S2O4 and the enzymatic reduction of NaBO3 or H2O2 by bovine liver catalase trapped in hydrated micelles of dioctylsulfosuccinate (AOT)/toluene were used as model systems. O2 titration with the above systems showed that air-saturated 1.6 M H2O/0.2 M AOT/toluene media contain seven times more O2 (1.4 mM) than aqueous solutions (0.2 mM). The measured Km values of catalase for NaBO3 and H2O2 in organic media were Kmov = 15 and 17 mM, respectively, whereas in aqueous buffer the values were 45 and 54 mM. In the toluene media, catalase activity increased with the W0 (H2O/AOT molar ratio) of the micellar preparation, reaching maximal activity at W0 = 10-12; under this condition, the catalytic center activity (Kp) of H2O2 was 7 x 10(6) min-1, similar to that obtained in the aqueous buffer (H2O2 = 7 x 10(6) min-1). It was found that the optimal pH for catalase in toluene media (pH 8.0) was shifted 1.0 unit compared to that in the aqueous buffer (pH 7.0). On the other hand, catalase was severely inhibited by NaN3 in both media. Thus, polarography based on the Clark oxygen electrode seems to be an easy, rapid, and sensitive technique for studying enzyme reactions consuming or evolving O2 in apolar media.     

The mechanism distinguishability problem in biochemical kinetics: the single-enzyme, single-substrate reaction as a case study

A theoretical analysis of the distinguishability problem of two rival models of the single enzyme-single substrate reaction, the Michaelis-Menten and Henri mechanisms, is presented. We also outline a general approach for analysing the structural indistinguishability between two mechanisms. The approach involves constructing, if possible, a smooth mapping between the two candidate models. Evans et al. [N.D. Evans, M.J. Chappell, M.J. Chapman, K.R. Godfrey, Structural indistinguishability between uncontrolled (autonomous) nonlinear analytic systems, Automatica 40 (2004) 1947-1953] have shown that if, in addition, either of the mechanisms satisfies a particular criterion then such a transformation always exists when the models are indistinguishable from their experimentally observable outputs. The approach is applied to the single enzyme-single substrate reaction mechanism. In principle, mechanisms can be distinguished using this analysis, but we show that our ability to distinguish mechanistic models depends both on the precise measurements made, and on our knowledge of the system prior to performing the kinetics experiments.     

Analytic solution to steady-state radial diffusion of a substrate with first-order reaction kinetics in the tissue of a Krogh's cylinder

It is often useful to calculate the concentration profile for a substrate undergoing reaction in the tissue surrounding a capillary. In this paper, we consider a model geometry consisting of a long straight cylinder of tissue surrounding a capillary. Substrate diffuses radially out of the capillary through the tissue, with consumption of substrate in the tissue directly proportional to substrate concentration (i.e., first-order reaction kinetics). The model is extended to include the case where a cylinder of necrotic tissue surrounds a metabolically active inner tissue cylinder. A simple analytic solution is derived, and concentration profiles are generated for various combinations of parameters. Compared to the case where substrate consumption is independent of concentration, this model predicts much more rapid depletion of substrate near the capillary interface. This can have significant implications for the calculation of the hypoxic fraction (e.g., tissue with pO(2)<0.5-5 mmHg) when tumor oxygenation is modeled. The model also permits calculation of the limiting substrate concentration for cell viability when the reaction rate constant is known and vice versa.     

Lecithin:cholesterol acyltransferase. Functional regions and a structural model of the enzyme

The amino acid sequence of human lecithin:cholesterol acyltransferase has been determined by degradation and alignment of peptides obtained from tryptic and staphylococcal digestions and the cleavage with cyanogen bromide and consisted of 416 amino acid residues. All of the tryptic peptides of lecithin:cholesterol acyltransferase were isolated and sequenced. Peptides resulting from digestion by staphylococcal protease, cyanogen bromide cleavage, or the combination of the two methods were employed to find overlapping segments. The N terminus of human lecithin:cholesterol acyltransferase was determined to be phenylalanine by sequencing the whole protein up to 40 residues while the C terminus was identified as glutamic acid through carboxypeptidase Y cleavage. Cys50 and Cys74 and Cys313 and Cys356 were identified as the two disulfide bridges while the free sulfhydryl groups were located at positions 31 and 184. The N-glycosylated sites of the protein were assigned to asparagines at positions 20, 84, 272, and 384. The active site of lecithin:cholesterol acyltransferase was identified as serine on position 181 according to its homology with other serine-type esterases which have a common structure of glycine-variable amino acid-active serine-variable amino acid-glycine (Gly-X-Ser-X-Gly) with the variable amino acids disrupting the homology. No long internal repeats or homologies with apolipoproteins were found. The secondary structure is consistent with the results of predictive algorithms. A simple model of the enzyme is proposed on the basis of available chemical data and predictive methods.     

Application of the principles of enzyme kinetics to clonal growth rate assays: an approach for delineating interactions among growth promoting agents.

The interaction of mitogenic factors on a single cell type and the comparative activity of a given factor in diverse cell types have been studied by applying the principles of Michaelis-Menten kinetics to clonal growth data. Such comparisons are facilitated by derivation of two parameters; Km mitogen, the mitogen concentration that gives half-maximal clonal growth and a theoretical maximal growth rate, RMAX T. Both parameters are analogous to the Km and VMAX as applied to enzymatic reactions. Use of these parameters permits meaningful comparisons between cells with different growth rates. Using kinetic analysis of dose-response data, we found that normal human epithelial cells require 200 times more fetal bovine serum protein (FBSP) than a malignant line to multiply at their respective half-maximal rates. Further, the Km FBSP of normal cells was reduced to that of the malignant line by the inclusion of growth factors (EGF or FGF, and hydrocortisone) in the medium. On the other hand, even though greater levels of serum were required when growth factors and hydrocortisone were not present, their inclusion did not alter RMAX T. Interactions between mitogenic factors were shown to be unidirectional. Although EGF reduced the Km FBSP, FBSP did not change the Km EGF. The same type of analysis revealed that hydrocortisone, which potentiated the mitogenic activity of EGF did not change the Km EGF. Kinetic analysis of cell growth should prove useful in studies on the relation between growth and tumor promotion as well as in the evaluation of growth-inhibiting chemotherapeutic agents.     

Normalization in the fitting of data by iterative methods. Application to tracer kinetics and enzyme kinetics

1. The normalization of biochemical data to weight them appropriately for parameter estimation is considered, with reference particularly to data from tracer kinetics and enzyme kinetics. If the data are in replicate, it is recommended that the sum of squared deviations for each experimental variable at each time or concentration point is divided by the local variance at that point. 2. If there is only one observation for each variable at each sampling point, normalization may still be required if the observations cover more than one order of magnitude, but there is no absolute criterion for judging the effect of the weighting that is produced. The goodness of fit that is produced by minimizing the weighted sum of squares of deviations must be judged subjectively. It is suggested that the goodness of fit may be regarded as satisfactory if the data points are distributed uniformly on either side of the fitted curve. A chi-square test may be used to decide whether the distribution is abnormal. The proportion of the residual variance associated with points on one or other side of the fitted curve may also be taken into account, because this gives an indication of the sensitivity of the residual variance to movement of the curve away from particular data points. These criteria for judging the effect of weighting are only valid if the model equation may reasonably be expected to apply to all the data points. 3. On this basis, normalizing by dividing the deviation for each data point by the experimental observation or by the equivalent value calculated by the model equation may both be shown to produce a consistent bias for numerically small observations, the former biasing the curve towards the smallest observations, the latter tending to produce a curve that is above the numerically smaller data points. It was found that dividing each deviation by the mean of observed and calculated variable appropriate to it produces a weighting that is fairly free from bias as judged by the criteria mentioned above. This normalization factor was tested on published data from both tracer kinetics and enzyme kinetics.