Enzyme kinetics, structural analysis and molecular modeling studies on a series of Schistosoma mansoni PNP inhibitors

The enzyme purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive molecular target for the development of novel drugs against schistosomiasis, a neglected tropical disease that affects about 200 million people worldwide. In the present work, enzyme kinetic studies were carried out in order to determine the potency and mechanism of inhibition of a series of SmPNP inhibitors. In addition to the biochemical investigations, crystallographic and molecular modeling studies revealed important molecular features for binding affinity towards the target enzyme, leading to the development of structure-activity relationships (SAR).

Single molecule simulation of diffusion and enzyme kinetics

This work presents a molecular-scale agent-based model for the simulation of enzymatic reactions at experimentally measured concentrations. The model incorporates stochasticity and spatial dependence, using diffusing and reacting particles with physical dimensions. We developed strategies to adjust and validate the enzymatic rates and diffusion coefficients to the information required by the computational agents, i.e., collision efficiency, interaction logic between agents, the time scale associated with interactions (e.g., kinetics), and agent velocity. Also, we tested the impact of molecular location (a source of biological noise) in the speed at which the reactions take place. Simulations were conducted for experimental data on the 2-hydroxymuconate tautomerase (EC 5.3.2.6, UniProt ID Q01468) and the Steroid Delta-isomerase (EC 5.3.3.1, UniProt ID P07445). Obtained results demonstrate that our approach is in accordance to existing experimental data and long-term biophysical and biochemical assumptions.

Efficient and accurate experimental design for enzyme kinetics: Bayesian studies reveal a systematic approach

In areas such as drug development, clinical diagnosis and biotechnology research, acquiring details about the kinetic parameters of enzymes is crucial. The correct design of an experiment is critical to collecting data suitable for analysis, modelling and deriving the correct information. As classical design methods are not targeted to the more complex kinetics being frequently studied, attention is needed to estimate parameters of such models with low variance. We demonstrate that a Bayesian approach (the use of prior knowledge) can produce major gains quantifiable in terms of information, productivity and accuracy of each experiment. Developing the use of Bayesian Utility functions, we have used a systematic method to identify the optimum experimental designs for a number of kinetic model data sets. This has enabled the identification of trends between kinetic model types, sets of design rules and the key conclusion that such designs should be based on some prior knowledge of K-M and/or the kinetic model. We suggest an optimal and iterative method for selecting features of the design such as the substrate range, number of measurements and choice of intermediate points. The final design collects data suitable for accurate modelling and analysis and minimises the error in the parameters estimated. (C) 2003 Elsevier Science B.V. All rights reserved.

Calorimetric demonstration of the potential of molecular crowding to emulate the effect of an allosteric activator on pyruvate kinase kinetics

A method based on isothermal calorimetry is described for the direct kinetic assay of pyruvate kinase. In agreement with earlier findings based on the standard coupled assay system for this enzyme in the presence of a fixed ADP concentration, the essentially rectangular hyperbolic dependence of initial velocity upon phosphoenolpyruvate concentration is rendered sigmoidal by the allosteric inhibitor phenylalanine. This effect of phenylalanine can be countered by including a high concentration of a space- filling osmolyte such as proline in the reaction mixtures. This investigation thus affords a dramatic example that illustrates the need to consider potential consequences of thermodynamic nonideality on the kinetics of enzyme reactions in crowded molecular environments such as the cell cytoplasm.

Further evidence for the reliance of catalysis by rabbit muscle pyruvate kinase upon isomerization of the ternary complex between enzyme and products

Isothermal calorimetry has been used to examine the effect of thermodynamic non-ideality on the kinetics of catalysis by rabbit muscle pyruvate kinase as the result of molecular crowding by inert cosolutes. The investigation, designed to detect substrate-mediated isomerization of pyruvate kinase, has revealed a 15% enhancement of maximal velocity by supplementation of reaction mixtures with 0.1 M proline, glycine or sorbitol. This effect of thermodynamic non-ideality implicates the existence of a substrate-induced conformational change that is governed by a minor volume decrease and a very small isomerization constant; and hence, substantiates earlier inferences that the rate-determining step in pyruvate kinase kinetics is isomerization of the ternary enzyme product complex rather than the release of products. (C) 2003 Elsevier Science B.V. All rights reserved.

Steady-state brain glucose transport kinetics re-evaluated with a four-state conformational model.

Glucose supply from blood to brain occurs through facilitative transporter proteins. A near linear relation between brain and plasma glucose has been experimentally determined and described by a reversible model of enzyme kinetics. A conformational four-state exchange model accounting for trans-acceleration and asymmetry of the carrier was included in a recently developed multi-compartmental model of glucose transport. Based on this model, we demonstrate that brain glucose (G(brain)) as function of plasma glucose (G(plasma)) can be described by a single analytical equation namely comprising three kinetic compartments: blood, endothelial cells and brain. Transport was described by four parameters: apparent half saturation constant K(t), apparent maximum rate constant T(max), glucose consumption rate CMR(glc), and the iso-inhibition constant K(ii) that suggests G(brain) as inhibitor of the isomerisation of the unloaded carrier. Previous published data, where G(brain) was quantified as a function of plasma glucose by either biochemical methods or NMR spectroscopy, were used to determine the aforementioned kinetic parameters. Glucose transport was characterized by K(t) ranging from 1.5 to 3.5 mM, T(max)/CMR(glc) from 4.6 to 5.6, and K(ii) from 51 to 149 mM. It was noteworthy that K(t) was on the order of a few mM, as previously determined from the reversible model. The conformational four-state exchange model of glucose transport into the brain includes both efflux and transport inhibition by G(brain), predicting that G(brain) eventually approaches a maximum concentration. However, since K(ii) largely exceeds G(plasma), iso-inhibition is unlikely to be of substantial importance for plasma glucose below 25 mM. As a consequence, the reversible model can account for most experimental observations under euglycaemia and moderate cases of hypo- and hyperglycaemia.

Exploring the enzymatic landscape: distribution and kinetics of hydrolytic enzymes in soil particle-size fractions

The location of extracellular enzymes within the soil architecture and their association with the various soil components affects their catalytic potential. A soil fractionation study was carried out to investigate: (a) the distribution of a range of hydrolytic enzymes involved in C, N and P transformations, (b) the effect of the location on their respective kinetics, (c) the effect of long-term N fertilizer management on enzyme distribution and kinetic parameters. Soil (silty clay loam) from grassland which had received 0 or 200 kg N ha(-1) yr(-1) was fractionated, and four particle-size fractions (> 200, 200-63, 63-2 and 0. 1-2 mum) were obtained by a combination of wet-sieving and centrifugation, after low-energy ultrasonication. All fractions were assayed for four carbohydrases (beta-cellobiohydrolase, N-acetyl-beta-glucosammidase, beta-glucosidase and beta-xylosidase), acid phosphatase and leucine-aminopeptidase using a microplate fluorimetric assay based on MUB-substrates. Enzyme kinetics (V-max and K-m) were estimated in three particle-size fractions and the unfractionated soil. The results showed that not all particle-size fractions were equally enzymatically active and that the distribution of enzymes between fractions depended on the enzyme. Carbohydrases predominated in the coarser fractions while phosphatase and leucine-aminopeptidase were predominant in the clay-size fraction. The Michaelis constant (K.) varied among fractions, indicating that the association of the same enzyme with different particle-size fractions affected its substrate affinity. The same values of Km were found in the same fractions from the soil under two contrasting fertilizer management regimes, indicating that the Michaelis constant was unaffected by soil changes caused by N fertilizer management. (C) 2004 Elsevier Ltd. All rights reserved.

The Water Effect on the Kinetics of the Bovine Liver Catalase

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Kinetics and crystal structure of human purine nucleoside phosphorylase in complex with 7-methyl-6-thio-guanosine

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and drugs that inhibit this enzyme may have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Here, we describe kinetics and crystal structure of human PNP in complex with 7-methyl-6-thio-guanosine, a synthetic substrate, which is largely used in activity assays. Analysis of the structure identifies different protein conformational changes upon ligand binding, and comparison of kinetic and structural data permits an understanding of the effects of atomic substitution on key positions of the synthetic substrate and their consequences to enzyme binding and catalysis. Such knowledge may be helpful in designing new PNP inhibitors. © 2005 Elsevier Inc. All rights reserved.

The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses

Tuberculosis remains as one of the main cause of mortality worldwide due to a single infectious agent, Mycobacterium tuberculosis. The aroK-encoded M. tuberculosis Shikimate Kinase (MtSK), shown to be essential for survival of bacilli, catalyzes the phosphoryl transfer from ATP to the carbon-3 hydroxyl group of shikimate (SKH), yielding shikimate-3-phosphate and ADP. Here we present purification to homogeneity, and oligomeric state determination of recombinant MtSK. Biochemical and biophysical data suggest that the chemical reaction catalyzed by monomeric MtSK follows a rapid-equilibrium random order of substrate binding, and ordered product release. Isothermal titration calorimetry (ITC) for binding of ligands to MtSK provided thermodynamic signatures of non-covalent interactions to each process. A comparison of steady-state kinetics parameters and equilibrium dissociation constant value determined by ITC showed that ATP binding does not increase the affinity of MtSK for SKH. We suggest that MtSK would more appropriately be described as an aroL-encoded type II shikimate kinase. Our manuscript also gives thermodynamic description of SKH binding to MtSK and data for the number of protons exchanged during this bimolecular interaction. The negative value for the change in constant pressure heat capacity (ΔCp) and molecular homology model building suggest a pronounced contribution of desolvation of non-polar groups upon binary complex formation. Thermodynamic parameters were deconvoluted into hydrophobic and vibrational contributions upon MtSK:SKH binary complex formation. Data for the number of protons exchanged during this bimolecular interaction are interpreted in light of a structural model to try to propose the likely amino acid side chains that are the proton donors to bulk solvent following MtSK:SKH complex formation. © 2013 Rosado et al.

Cyclodextrin Production by Bacillus lehensis Isolated from Cassava Starch: Characterisation of a Novel Enzyme

The properties of a previously unknown enzyme, denominated cyclodextrin glycosyltransferase, produced from Bacillus lehensis, were evaluated using affinity chromatography for protein purification. Enzyme characteristics (optimum pH and temperature; pH and temperature stability), the influence of substances on the enzyme activity, enzyme kinetics, and cyclodextrin production were analysed. Cyclodextrin glycosyltransferase was purified up to 320.74-fold by affinity chromatography using beta-cyclodextrin as the binder and it exhibited 8.71% activity recovery. This enzyme is a monomer with a molecular weight of 81.27 kDa, as estimated by SDS-PAGE. Optimum temperature and pH for cydodextrin glycosyltransferase were 55 degrees C and 8.0, respectively. The Michaelis-Menten constant was 8.62 g/l during maximum velocity of 0.858 g/l.h.

Modeling physiological processes in plankton on enzyme kinetic principles

[EN] Many ecologically important chemical transformations in the ocean are controlled by biochemical enzyme reactions in plankton. Nitrogenase regulates the transformation of N2 to ammonium in some cyanobacteria and serves as the entryway for N2 into the ocean biosphere. Nitrate reductase controls the reduction of NO3 to NO2 and hence new production in phytoplankton. The respiratory electron transfer system in all organisms links the carbon oxidation reactions of intermediary metabolism with the reduction of oxygen in respiration. Rubisco controls the fixation of CO2 into organic matter in phytoplankton and thus is the major entry point of carbon into the oceanic biosphere. In addition to these, there are the enzymes that control CO2 production, NH4 excretion and the fluxes of phosphate. Some of these enzymes have been recognized and researched by marine scientists in the last thirty years. However, until recently the kinetic principles of enzyme control have not been exploited to formulate accurate mathematical equations of the controlling physiological expressions. Were such expressions available they would increase our power to predict the rates of chemical transformations in the extracellular environment of microbial populations whether this extracellular environment is culture media or the ocean. Here we formulate from the principles of bisubstrate enzyme kinetics, mathematical expressions for the processes of NO3 reduction, O2 consumption, N2 fixation, total nitrogen uptake.

A windows program for the derivation of steady-state equations in enzyme systems

Despite the number of computer-assisted methods described for the derivation of steady-state equations of enzyme systems, most of them are focused on strict steady-state conditions or are not able to solve complex reaction mechanisms. Moreover, many of them are based on computer programs that are either not readily available or have limitations. We present here a computer program called WinStes, which derives equations for both strict steady-state systems and those with the assumption of rapid equilibrium, for branched or unbranched mechanisms, containing both reversible and irreversible conversion steps. It solves reaction mechanisms involving up to 255 enzyme species, connected by up to 255 conversion steps. The program provides all the advantages of the Windows programs, such as a user-friendly graphical interface, and has a short computation time. WinStes is available free of charge on request from the authors. (c) 2006 Elsevier Inc. All rights reserved.

Sequence-related structural DNA anomalies affect restriction enzyme activity and DNA polymerase I binding

DNA serves as a target molecule for several types of enzymes and may assume a wide variety of structural motifs depending upon the local sequence. The BssHII restriction site (GC)3 resides in a 9bp region of alternating pyrimidine and purine residues within the &phis;X174 genome. Such sequences are known to demonstrate non-canonical helical behavior under the appropriate conditions. The kinetics of BssHII cleavage was investigated in supercoiled and linear plasmid DNA, and in a 323bp DNA fragment obtained via amplification of &phis;X174. The rate of enzyme cleavage was enhanced in the supercoiled form and in the presence of 50μM cobalt hexamine. Similarly, cobalt hexamine was also found to enhance TaqI activity directly adjacent to the (GC)3 region. ^ Initial DNA polymerase I binding studies (including a gel mobility shift assay and a protection assay) indicated a notable interaction between DNA polymerase I and the BssHII site. An in-depth study revealed that equilibrium binding of DNA polymerase I to the T7 RNA polymerase promoter was comparable to that of the (GC)3 site, however the strongest interaction was observed with a cruciform containing region. Increasing the ionic strength of the solution environment, including the addition of DNA polymerase I reaction buffer significantly decreased the equilibrium dissociation constant values. ^ It is suggested that the region within or around the BssHII site experiences a conformational change generating a novel structure under the influence of supercoiled tension or 50μM cobalt hexamine. It is proposed that this transition may enhance enzyme activity and binding by providing an initial enzyme-docking site—the rate-limiting step in restriction enzyme kinetics. The high binding potential of DNA polymerase I for each of the motifs described, is hypothesized to be due to recognition of the structural DNA anomalies by the 3′–5′ exonuclease domain. ^

Functional analysis, using in vitro mutagenesis, of amino acids located in the phenylalanine hydroxylase active site

The 3-dimensionaI structure determination of rat phenylalanine hydroxylase (PAH) has identified potentially important amino acids lining the active site cleft with the majority of these having hydrophobic side-chains including several with aromatic side chains. Here we have analyzed the effect on rat PAH enzyme kinetics of in vitro mutagenesis of a number of these amino acids lining the PAH active site. Mutation of F299, Y324, F331, and Y343 caused a significant decrease in enzyme activity but no change in the K-m for substrate or cofactor. me conclude that these aromatic residues are essential for activity but are not significantly involved in binding of the substrate or cofactor. in contrast the PAH mutant, S349T, showed an 18-fold increase in K-m for phenylalanine, showing the first functional evidence that this residue was binding at or near the phenylalanine binding site. This confirms the recently published model for the binding of phenylalanine to the PAH active site that postulated S349 interacts with the amino group on the main chain of the phenylalanine molecule. This result differs with that found for the equivalent mutation (S395T), in the closely related tyrosine hydroxylase, which had no effect on substrate K-m, showing that while the architecture of the two active sites are very similar the amino acids that bind to the respective substrates are different. (C) 2000 Academic Press.