Why metabolic enzymes are essential or nonessential for growth of Escherichia coli K12 on glucose

The genes encoding metabolic enzymes involved in glucose metabolism, the TCA cycle, and biosynthesis of amino acids, purines, pyrimidines, and cofactors would be expected to be essential for growth of Escherichia coli on glucose because the cells must synthesize all of the building blocks for cellular macromolecules. Surprisingly, 80 of 227 of these genes are not essential. Analysis of why these genes are not essential provides insights into the metabolic sophistication of E. coli and into the evolutionary pressures that have shaped its physiology. Alternative routes enabled by interconnecting pathways can allow a defective step to be bypassed. Isozymes, alternative enzymes, broad-specificity enzymes, and multifunctional enzymes can often substitute for a missing enzyme. We expect that the apparent redundancy in these metabolic pathways has arisen due to the need for E. coli to survive in a variety of habitats and therefore to have a metabolism that allows optimal exploitation of varying environmental resources and synthesis of small molecules when they cannot be obtained from the environment.     

Corrected equations for calculation of constants in enzyme inhibition and activation

Equations for calculation of the constants of biparametrical types of enzyme inhibition and activation were obtained that take into account a ratio of the lengths of L vector projections representing such reactions in the three-dimensional K (m)V I coordinate system. This allows higher accuracy of calculation and is more correct for comparison of these constants. Examples of data analysis of enzyme inhibition and activation by using the traditional equations (they do not take into account the lengths of vector projections) and corrected ones (they take into account the lengths of vector projections) are given. The corrected and traditional equations are used for calculation of the constants of biparametrical types of enzyme inhibition and activation.     

Production of bacteriolytic enzymes as a tool for characterizing enterococci

Bacteriolytic enzymes secreted by log-phase cultures of enterococci ( Enterococcus faecalis, Ent. faecium, Ent. durans, Ent. hirae, Ent. casseliflavus, Ent. avium, Ent. mundtii ) were analysed by means of a zymogram technique to resolve activities according to the size of their polypeptide component and their specificities towards different substrates. Heterogeneous patterns of lytic activity were observed with different species. For each test substrate, homogeneous patterns of lytic activities were observed with strains of the same species, except for Ent. faecalis strains, which showed heterogeneous lytic patterns even towards the same substrate, and could be divided into at least four different groups according to their lytic pattern. No lytic activity was common to all strains tested. Results of zymogram analysis of Enterococcus bacteriolytic enzymes were consistent with current knowledge on enterococcal taxonomy, indicating that this analytical approach may be a useful tool for fine-tuned characterization of different enterococcal strains.     

Simplified equations for the distribution of chloride in body water

According to the theory of Boyle and Conway, chloride leaves the extracellular fluid to enter cells, particularly muscle, when the plasma potassium concentrationK rises. Simplified equations are presented to describe in terms ofK the distribution of chloride expected when the quantity of the saltKp in the body fluids changes in health and disease.     

Platinum speciation used for elucidating activation or inhibition of Pt-containing anti-cancer drugs

This article reviews approaches on platinum speciation with respect to Pt drugs in anti-cancer therapies. The paper starts with the introduction of available platinum-based drugs and describes their assumed principle of action. It is now generally accepted that these Pt complexes exhibit their therapeutic action by coordination to DNA which leads to bending of the DNA structure and to an inhibition of the DNA polymerase progression. But dose-limiting side effects, including nephrotoxicity as well as resistance to some of these Pt compounds, are still a major problem. Platinum speciation moved increasingly into the focus of interest when it became clear that (1) the active drugs were the hydrolyzation products rather than the originally administered ones and (2) that the parallel formation of inactive Pt–protein complexes, which additionally reduce the efficacy of Pt anti-tumor agents, compete with the formation of the cytotoxic Pt-DNA lesions. Speciation analysis methods were employed based on chromatography or capillary electrophoresis respectively, each coupled to inductively coupled plasma (ICP)-mass spectrometry (MS) or electrospray ionization (ESI)-MS.The paper describes these Pt-speciation investigations, which started with exploring hydrolyzation kinetics in aqueous solutions. These experiments were followed by the speciation investigations in model solutions containing proteins or other sulphur-containing ligands, which could also be responsible for deactivation of the Pt agent in vivo. The experiments improved the understanding of the metabolite form, by which the metal complex enters the tumor cells, and whether and how this metabolized complex is already inactivated at this time. As an example, reaction kinetics of cisplatin (cis-[diamminedichloroplatinum(II)]) with albumin, transferrin, myoglobin, ubiquitin, and metallothionein were investigated and reaction products were speciated.Finally, Pt-speciation in serum of medicated cancer patients was conducted by several research groups, which are outlined in the Section “Investigations in serum”.The section “Investigations in urine of cancer treated patients” deals with speciation experiments on the Pt-metabolites excreted by the organism. By these means an assessment of the in vivo metabolism of Pt-drugs may be possible. Finally, the development of new anti-cancer metallodrugs needs the respective analytical techniques reported in the last section of the paper.     

Basic models for differential inhibition of enzymes

The possible preferential action exerted by an inhibitor on the transformation of one of two agonist substrates catalyzed by the same enzyme has recently been reported in studies on aldose reductase inhibition. This event was defined as “intra-site differential inhibition” and the molecules able to exert this action as “differential inhibitors”. This work presents some basic kinetic models describing differential inhibition. Using a simple analytic approach, the results show that differential inhibition can occur through either competitive or mixed type inhibition in which the inhibitor prevalently targets the free enzyme. The results may help in selecting molecules whose differential inhibitory action could be advantageous in controlling the activity of enzymes acting on more than one substrate.     

Simplified method for the computation of parameters of power-law rate equations from time-series

Modeling biological processes from time-series data is a resourceful procedure which has received much attention in the literature. For models established in the context of non-linear differential equations, parameter-dependent phenomenological tentative response functions are tested by comparing would-be solutions of those models to the experimental time-series. Those values of the parameters for which a tested solution is a best fit are then retained. It is done with the help of some appropriate optimization algorithm which simplifies the searching procedure within the range of variability of the parameters that are to be estimated. The procedure works well in problems with a small number of adjustable parameters or/and with narrow searching ranges. However, it may start to be problematic for models with a large number of problem parameters inasmuch as convergence to the best fit is not necessarily ensured. In this case, a reduction in size of the parameter estimation problem must be undertaken. We presently address this issue by proposing a systematic procedure that does so in problems in which the system's response to a sufficiently small pulse perturbation of steady-state can be obtained. The response is then assumed to be a solution of the linearized equations, the Jacobian of which can be retrieved by a simple multilinear regression. The calculated n(2) Jacobian entries provide as many relationships among problem parameters, thus cutting substantially the size of the starting problem. After this preliminary treatment is applied, only (kappa-n(2)) of the initial kappa adjustable parameters are left for evaluation by means of a non-linear optimization procedure. The benefits of the present variant are both in economy of computation and in accuracy in determining the parameter values. The performance of the method is established under different circumstances. It is illustrated in the context of power-law rates, although this does not preclude its applicability to more general functional responses.     

Adrenal cortical 11 beta-hydroxylase and side-chain cleavage enzymes. Requirement for the A- or B-pyridyl ring in metyrapone for inhibition

The adrenal cortical enzyme systems, 11 beta-hydroxylase, P-450 11 beta, and the side-chain cleavage complex, P-450 scc, differ only in their cytochrome P-450s. Structural modifications of metyrapone, an inhibitor of cytochrome P-450 enzyme systems, have been made to determine the requirement for the A- or B-pyridyl ring for inhibition of P-45011 beta and P-450 scc activities. Three new analogs of metyrapone (A-phenylmetyrapone, B-phenylmetyrapone and diphenylmetyrapone) were synthesized and evaluated as inhibitors using a crude, defatted bovine adrenal cortical mitochondrial preparation. Characterization of the mitochondrial preparation demonstrated: enhancement of both activities by the addition of 15.0 microM adrenodoxin, the addition of 1% ethanol decreased both activities less than 10%, and the apparent Km of deoxycorticosterone for P-45011 beta was 6.8 microM and the apparent Km of cholesterol for P-450 scc was 21.6 microM. Inhibition of P-45011 beta and P-450 scc activities with these compounds demonstrated: the B-pyridyl ring of metyrapone is required for inhibition of both activities whereas requirement for the A-ring is less stringent, and the four metyrapone analogs were more selective inhibitors of P-45011 beta activity. These studies suggest that the A-phenyl metyrapone analog is a good candidate for further development of a selective adrenocortical radiopharmaceutical.     

Substrate inhibition or activation kinetics of the beta-galactosidase from the extreme thermoacidophile archaebacterium Caldariella acidophila

The kinetics of the hydrolysis of p-nitrophenyl-beta-D-galactopyranoside (pNPG) by a thermophile, beta-galactosidase, was studied at different temperatures. This enzyme was isolated from the thermophilic microorganism archaebacterium Caldariella acidophila. The hydrolysis of pNPG by beta-galactosidase does not follow Michaelis-Menten law. This enzyme is inhibited by excess substrate at low temperatures and it is activated by excess substrate at high temperatures. A minimum mechanistic model is proposed to explain the behaviour. This model assumes the binding of an additional substrate molecule on the glycosidyl enzyme intermediate. This model is in good agreement with the postulated mechanism for beta-galactosidase from Escherichia coli. The kinetic parameters are calculated at six different temperatures.     

Structural determinants for activation or inhibition of ryanodine receptors by basic residues in the dihydropyridine receptor II-III loop

The structures of peptide A, and six other 7-20 amino acid peptides corresponding to sequences in the A region (Thr671- Leu690) of the skeletal muscle dihydropyridine receptor II-III loop have been examined, and are correlated with the ability of the peptides to activate or inhibit skeletal ryanodine receptor calcium release channels. The peptides adopted either random coil or nascent helix-like structures, which depended upon the polarity of the terminal residues as well as the presence and ionisation state of two glutamate residues. Enhanced activation of Ca2+ release from sarcoplasmic reticulum, and activation of current flow through single ryanodine receptor channels (at -40 mV), was seen with peptides containing the basic residues 681Arg Lys Arg Arg Lys685, and was strongest when the residues were a part of an alpha-helix. Inhibition of channels (at +40 mV) was also seen with peptides containing the five positively charged residues, but was not enhanced in helical peptides. These results confirm the hypothesis that activation of ryanodine receptor channels by the II-III loop peptides requires both the basic residues and their participation in helical structure, and show for the first time that inhibition requires the basic residues, but is not structure-dependent. These findings imply that activation and inhibition result from peptide binding to separate sites on the ryanodine receptor.     

An optimization framework for identifying reaction activation/inhibition or elimination candidates for overproduction in microbial systems

We introduce a computational framework termed OptReg that determines the optimal reaction activations/inhibitions and eliminations for targeted biochemical production. A reaction is deemed up- or downregulated if it is constrained to assume flux values significantly above or below its steady-state before the genetic manipulations. The developed framework is demonstrated by studying the overproduction of ethanol in Escherichia coli. Computational results reveal the existence of synergism between reaction deletions and modulations implying that the simultaneous application of both types of genetic manipulations yields the most promising results. For example, the downregulation of phosphoglucomutase in conjunction with the deletion of oxygen uptake and pyruvate formate lyase yields 99.8% of the maximum theoretical ethanol yield. Conceptually, the proposed strategies redirect both the carbon flux as well as the cofactors to enhance ethanol production in the network. The OptReg framework is a versatile tool for strain design which allows for a broad array of genetic manipulations.     

The inhibition of enzymes by beryllium

1. The action of beryllium on the following enzymes has been examined: alkaline phosphatase (Escherichia coli and kidney), acid phosphatase, phosphoprotein phosphatase, apyrase (potato), adenosine triphosphatase (liver nuclei, liver mitochondria, brain microsomes), glucose 6-phosphatase, polysaccharide phosphorylases a and b, phosphoglucomutase, hexokinase, phosphoglyceromutase, ribonuclease, A-esterase (rabbit serum), cholinesterase (horse serum), chymotrypsin. Alkaline phosphatase and phosphoglucomutase are inhibited by 1mum-beryllium sulphate whereas the other enzymes are largely unaffected by 1mm-beryllium sulphate. 2. Possible mechanisms for the inhibition of phosphoglucomutase and alkaline phosphatase are discussed.