Steady-state kinetic behaviour of two- or n-enzyme systems made of free sequential enzymes involved in a metabolic pathway

The overall rate of functioning of a set of free sequential enzymes of the Michaelis–Menten type involved in a metabolic pathway has been computed as a function of the concentration of the initial substrate under steady-state conditions. Curves monotonically increasing up to a saturation plateau have been obtained in all cases. The shape of these curves is sometimes, but not usually, close to that of a hyperbola. Cases exist in which the overall rate of reaction becomes quasi proportional to the concentration of initial substrate almost up to the saturation plateau, which never occurs with individual enzymes. Increasing the number of enzymes sequentially involved in a metabolic pathway does not seem to generate any particularly original behaviour compared with that of two-enzyme systems. To cite this article: G. Legent et al., C. R. Biologies 329 (2006).     

Steady-state kinetic behaviour of functioning-dependent structures

A fundamental problem in biochemistry is that of the nature of the coordination between and within metabolic and signalling pathways. It is conceivable that this coordination might be assured by what we term functioning-dependent structures (FDSs), namely those assemblies of proteins that associate with one another when performing tasks and that disassociate when no longer performing them. To investigate a role in coordination for FDSs, we have studied numerically the steady-state kinetics of a model system of two sequential monomeric enzymes, E(1) and E(2). Our calculations show that such FDSs can display kinetic properties that the individual enzymes cannot. These include the full range of basic input/output characteristics found in electronic circuits such as linearity, invariance, pulsing and switching. Hence, FDSs can generate kinetics that might regulate and coordinate metabolism and signalling. Finally, we suggest that the occurrence of terms representative of the assembly and disassembly of FDSs in the classical expression of the density of entropy production are characteristic of living systems.     

Reversible transdominant inhibition of a metabolic pathway. In vivo evidence of interaction between two sequential tricarboxylic acid cycle enzymes in yeast

The enzymes of the Krebs tricarboxylic acid cycle in mitochondria are proposed to form a supramolecular complex, in which there is channeling of intermediates between enzyme active sites. While interactions have been demonstrated in vitro between most of the sequential tricarboxylic acid cycle enzymes, no direct evidence has been obtained in vivo for such interactions. We have isolated, in the Saccharomyces cerevisiae gene encoding the tricarboxylic acid cycle enzyme citrate synthase Cit1p, an "assembly mutation," i.e. a mutation that causes a tricarboxylic acid cycle deficiency without affecting the citrate synthase activity. We have shown that a 15-amino acid peptide from wild type Cit1p encompassing the mutation point inhibits the tricarboxylic acid cycle in a dominant manner, and that the inhibitory phenotype is overcome by a co-overexpression of Mdh1p, the mitochondrial malate dehydrogenase. These data provide the first direct in vivo evidence of interaction between two sequential tricarboxylic acid cycle enzymes, Cit1p and Mdh1p, and indicate that the characterization of assembly mutations by the reversible transdominant inhibition method may be a powerful way to study multienzyme complexes in their physiological context.     

Clustering of sequential enzymes in the glycolytic pathway and the citric acid cycle

In recent years, evidence has been accumulating that metabolic pathways are organized in vivo as multienzyme clusters. Affinity electrophoresis proves to be an attractive in vitro method to further evidence specific associations between purified consecutive enzymes from the glycolytic pathway on the one hand, and from the citric acid cycle on the other hand. Our results support the hypothesis of cluster formation between the glycolytic enzymes aldolase, glyceraldehydephosphate dehydrogenase, and triosephosphate isomerase, and between the cycle enzymes fumarase, malate dehydrogenase, and citrate synthase. A model is presented to explain the possibility of regulation of the citric acid cycle by varying enzyme-enzyme associations between the latter three enzymes, in response to changing local intramitochondrial ATP/ADP ratios.     

Steady-state kinetics of bifunctional enzymes. Taking into account kinetic hierarchy of fast and slow catalytic cycles in a generalized model

A steady-state approximation of the generalized two-dimensional model of a bifunctional enzyme catalyzing independent proceeding of two one-pathway reactions is considered in a case of mutual influence of the active sites. Coexistence of fast and slow catalytic cycles in the reaction mechanism is analyzed. Conditions when the hierarchy of fast and slow catalytic cycles allows simplification of a two-dimensional model and its reduction to the one-dimensional cyclic schemes were determined. Kinetic equations describing these simplified schemes are presented.     

Molecular adaptation of enzymes, metabolic systems and transport systems in halophilic bacteria

Abstract A review is presented of the special properties and behaviour of enzymes, ribosomes, metabolic systems, protein turnover and active transport systems that are associated with the ability of halophilic archaebacteria and eubacteria to grow in different salt concentrations.     

The theoretical analysis of kinetic behaviour of “hysteretic” allosteric enzymes V. Relaxation kinetics of dissociating enzyme systems

The expressions for relaxation time as a function of enzyme and specific ligand concentration are deduced for dissociating enzyme system 2p ? P (P is enzyme oligomer which is able to dissociate reversibly forming two identical halves p). It is assumed that ligand binding sites are equivalent and independent in each oligomeric enzyme form and the equilibrium between oligomeric forms develops rather slowly in comparison with the rate of the binding of the ligand. The kinetics of relaxation of the dissociating enzyme system 2p ? P with progressive change of the rate constants for association of oligomeric form p has been analysed in graphic form. The situations when one of the oligomeric enzyme forms is not able to bind the ligand are also considered. The principles of the analysis of relaxation kinetics of dissociating enzyme systems 2p ? P are discussed.     

Development of a multicomponent kinetic assay of the early enzymes in the Campylobacter jejuni N-linked glycosylation pathway

The human pathogen Campylobacter jejuni possesses a general N-linked glycosylation system that is known to play a role in pathogenicity; however, a detailed understanding of this role remains elusive. A considerable hindrance to studying bacterial N-glycosylation in vivo is the absence of small molecule inhibitors to reversibly control the process. This report describes a pathway-screening assay that targets the early enzymes of C. jejuni N-glycan biosynthesis that would enable identification of inhibitors to the first four steps in the pathway. The assay includes PglF, PglE, PglD, PglC, and PglA; the enzymes involved in the biosynthesis of an undecaprenyl diphosphate-linked disaccharide and monitors the transfer of [3H]GalNAc from the hydrophilic UDP-linked carrier to the lipophilic UndPP-diNAcBac (2,4-diacetamido-2,4,6-trideoxyglucose). The optimized assay has a Z'-factor calculated to be 0.77, indicating a robust assay suitable for screening. The diacylglycerol kinase from Streptococcus mutans, which provides a convenient method for phosphorylating undecaprenol, has been included in a modified version of the assay thereby allowing the screen to be conducted with entirely commercially available substrates.     

Regulated expression systems for the development of whole-cell biocatalysts expressing oxidative enzymes in a sequential manner

This work reports the preparation of two recombinant strains each containing two enzymatic activities mutually expressed through regulated systems for production of functionalized epoxides in one-pot reactions. One strain was Pseudomonas putida PaW340, containing the gene coding for styrene monooxygenase (SMO) from Pseudomonas fluorescens ST under the auto-inducing Ptou promoter and the TouR regulator of Pseudomonas sp. OX1 and the gene coding for naphthalene dihydrodiol dehydrogenase (NDDH) from P. fluorescens N3 under the Ptac promoter inducible by IPTG. The second strain was Escherichia coli JM109, in which the expression of SMO was under the control of the Pnah promoter and the NahR regulator of P. fluorescens N3 inducible by salicylate, while the gene expressing NDDH was under the control of the Plac promoter inducible by IPTG. SMO and NDDH activities were tested in bioconversion experiments using cinnamyl alcohol as reference substrate. The application that we selected is one example of the sequential use of the two enzymatic activities which require a temporal control of the expression of both genes.     

Drug target validation of the trypanothione pathway enzymes through metabolic modelling

A kinetic model of trypanothione [T(SH)(2)] metabolism in Trypanosoma cruzi was constructed based on enzyme kinetic parameters determined under near-physiological conditions (including glutathione synthetase), and the enzyme activities, metabolite concentrations and fluxes determined in the parasite under control and oxidizing conditions. The pathway structure is characterized by a T(SH)(2) synthetic module of low flux and low catalytic capacity, and another more catalytically efficient T(SH)(2) -dependent antioxidant/regenerating module. The model allowed quantification of the contribution of each enzyme to the control of T(SH)(2) synthesis and concentration (flux control and concentration control coefficients, respectively). The main control of flux was exerted by γ-glutamylcysteine synthetase (γECS) and trypanothione synthetase (TryS) (control coefficients of 0.58-0.7 and 0.49-0.58, respectively), followed by spermidine transport (0.24); negligible flux controls by trypantothione reductase (TryR) and the T(SH)(2)-dependent antioxidant machinery were determined. The concentration of reduced T(SH)(2) was controlled by TryR (0.98) and oxidative stress (-0.99); however, γECS and TryS also exerted control on the cellular level of T(SH(2)) when they were inhibited by more than 70%. The model predicted that in order to diminish the T(SH)(2) synthesis flux by 50%, it is necessary to inhibit γECS or TryS by 58 or 63%, respectively, or both by 50%, whereas more than 98% inhibition was required for TryR. Hence, simultaneous and moderate inhibition of γECS and TryS appears to be a promising multi-target therapeutic strategy. In contrast, use of highly potent and specific inhibitors for TryR and the antioxidant machinery is necessary to affect the antioxidant capabilities of the parasites.     

A general method for generation and analysis of defined mutations in enzymes involved in a tetrahydrofolate-interconversion pathway

In eukaryotes, 10-formyltetrahydrofolate (THF) synthetase, 5,10-methenyl-THF cyclohydrolase and 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. These reactions are generally catalyzed by three separate monofunctional enzymes in prokaryotic cells. In this report a general method for the generation, detection and analysis of specific mutations affecting the catalytic activity of any of the reactions catalyzed by C1-THF synthase or its monofunctional counterparts is described. The method relies on plasmid-borne expression of genes in strains of the yeast Saccharomyces cerevisiae that are missing one or more of the activities of C1-THF synthase. Specific segments of the gene are subjected in vitro to random mutagenesis, the mutant genes expressed in yeast and screened by phenotype for inactivating mutations. Plasmids encoding mutant enzymes are recovered for sequence analysis. One-step purification of C1-THF synthase from the yeast expression system is demonstrated. The feasibility and versatility of the method is shown with the yeast ADE3 gene encoding the cytoplasmic C1-THF synthase and the gene encoding the monofunctional 10-formyl-THF synthetase from Clostridium acidiurici.     

Activities of enzymes involved in the phenylpropanoid pathway in constitutively salicylic acid-producing tobacco plants

Chorismate mutase (CM, EC 5.4.99.5), phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and chalcone synthase (CHS, EC 2.3.1.74) activities were studied in constitutive salicylic acid-producing (CSA) tobacco plants in relation to the accumulation of flavonoids and chlorogenic acid. The CM, PAL and CHS activities in CSA-tobacco (Nicotiana tabacum cv. Samsun NN) plants were lower than in non-transgenic tobacco plants. Flavonoid and chlorogenic acid accumulation was suppressed in CSA-tobacco plants compared to those of non-transgenic tobacco plants.     

The genes and enzymes of the carotenoid metabolic pathway in Vitis vinifera L

ABSTRACT: BACKGROUND: Carotenoids are a heterogeneous group of plant isoprenoids primarily involved inphotosynthesis. In plants the cleavage of carotenoids leads to the formation of thephytohormones abscisic acid and strigolactone, and C13-norisoprenoids involved in thecharacteristic flavour and aroma compounds in flowers and fruits and are of specificimportance in the varietal character of grapes and wine. This work extends the previousreports of carotenoid gene expression and photosynthetic pigment analysis by providing anup-to-date pathway analysis and an important framework for the analysis of carotenoidmetabolic pathways in grapevine. RESULTS: Comparative genomics was used to identify 42 genes putatively involved in carotenoidbiosynthesis/catabolism in grapevine. The genes are distributed on 16 of the 19 chromosomesand have been localised to the physical map of the heterozygous ENTAV115 grapevinesequence. Nine of the genes occur as single copies whereas the rest of the carotenoidbiosynthetic genes have more than one paralogue. The cDNA copies of eleven correspondinggenes from Vitis vinifera L. cv. Pinotage were characterised, and four where shown to befunctional. Microarrays provided expression profiles of 39 accessions in the metabolicpathway during three berry developmental stages in Sauvignon blanc, whereas an optimisedHPLC analysis provided the concentrations of individual carotenoids. This provides evidenceof the functioning of the lutein epoxide cycle and their respective genes in grapevine.Similarly, orthologues of genes leading to the formation of strigolactone involved in shootbranching inhibition were identified: CCD7, CCD8 and MAX1. Moreover, the isoformstypically have different expression patterns, confirming the complex regulation of thepathway. Of particular interest is the expression pattern of the three VvNCEDs: Our resultssupport previous findings that VvNCED3 is likely the isoform linked to ABA content inberries. CONCLUSIONS: The carotenoid biosynthetic pathway is well characterised, and the genes and enzymes havebeen studied in a number of plants. The study of the 42 carotenoid pathway genes ofgrapevine showed that they share a high degree of similarity with other eudicots. Expressionand pigment profiling of developing berries provided insights into the most completegrapevine carotenoid pathway representation. This study represents an important referencestudy for further characterisation of carotenoid biosynthesis and catabolism in grapevine.