Substrate channeling.

Substrate channeling is the process in which the intermediate produced by one enzyme is transferred to the next enzyme without complete mixing with the bulk phase. This process is equivalent to a microcompartmentation of the intermediate, although classic diffusion occurs simultaneously to varying extents in many of these cases. This microcompartmentation and other factors of channeling provide many potential biological advantages. Extensive examples of channeling can be found in the cited reviews. The choice of methods to detect and characterize substrate channeling depends extensively on the type of enzyme associations involved, the constants of the system, and, to some extent, the mechanism of channeling. Thus it is important to distinguish stable, dynamic, and catalytically induced enzyme associations as well as recognize different mechanisms of substrate channeling. We discuss the principles, experimental details, and limitations and precautions of five rather general methods. These use measurements of transient times, isotope dilution or enhancement, competing reaction effects, enzyme buffering kinetics, and transient-state kinetics. These encompass methods applicable to studies in vitro, in situ, and in vivo. None of these methods is applicable to all systems. They are also susceptible to artifacts without proper attention to precautions. Transient-state kinetic methods clearly excel in elucidating molecular mechanisms of channeling. However, they are often not the best method for initial detection and characterization of the process and they are not applicable to many complex systems. Several other methods that have been successful in indicating substrate channeling are briefly described.     

Substrate channeling of oxalacetate in solid-state complexes of malate dehydrogenase and citrate synthase

Current evidence suggests that mitochondrial matrix enzymes exist in solid-state, multienzyme complexes in vivo. Addition of polyethylene glycol to a solution containing malate dehydrogenase and citrate synthase generates such a solid-state, enzyme complex in vitro at enzyme concentrations permitting kinetic measurements. Suspensions of the isolated, solid-state, hetero-complex of these enzymes were used to study the coupled reactions of citrate synthesis from malate, NAD, and CoASAc. The particles appear to be about 1 microgram in diameter. Considering the ratio of enzyme to oxalacetate molecules in or at the surface of the solid-state particles, one would expect oxalacetate to be converted to citrate within a few molecular distances of the site of oxalacetate generation. This model of "substrate channeling" (or alternatively a direct transfer of oxalacetate between enzymes) is supported by experiments with excess aspartate aminotransferase and glutamate added to the solution phase to give a reaction competing with the synthase for bulk phase oxalacetate. Quantities of aminotransferase that reduce the citrate reaction rate with soluble dehydrogenase and synthase by 90% do not significantly affect rates with comparable amounts of the dehydrogenase-synthase complex. We suggest that similar substrate channeling can occur in vivo and discuss the possible advantages provided thereby.     

Substrate channeling of NADH and binding of dehydrogenases to complex I

The binding of porcine heart mitochondrial malate dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase to bovine heart NADH:ubiquinone oxidoreductase (complex I), but not that of bovine heart alpha-ketoglutarate dehydrogenase complex, is virtually abolished by 0.1 mM NADH. The malate dehydrogenase and beta-hydroxyacyl-CoA enzymes compete in part for the same binding site(s) on complex I as do the malate dehydrogenase and alpha-ketoglutarate dehydrogenase complex enzymes. Associations between mitochondrial malate dehydrogenase and bovine serum albumin were observed. Subtle convection artifacts in short-time centrifugation tests of enzyme association with the Beckman Airfuge are described. Substrate channeling of NADH from both the mitochondrial and cytoplasmic malate dehydrogenase isozymes to complex I and reduction of ubiquinone-1 were shown to occur in vitro by transient enzyme-enzyme complex formation. Excess apoenzyme causes little inhibition of the substrate channeling reaction with both malate dehydrogenase isozymes in spite of tighter equilibrium binding than the holoenzyme to complex I. This substrate channeling could, in principle, provide a dynamic microcompartmentation of mitochondrial NADH.     

Patterns of indirect protein interactions suggest a spatial organization to metabolism

It has long been believed that cells organize their cytoplasm so as to efficiently channel metabolites between sequential enzymes. This metabolic channeling has the potential to yield higher metabolic fluxes as well as better regulatory control over metabolism. One mechanism for achieving such channeling is to ensure that sequential enzymes in a pathway are physically close to each other in the cell. We present evidence that indirect protein interactions between related enzymes represent a global mechanism for achieving metabolic channeling; the intuition being that protein interactions between enzymes and non-enzymatic mediator proteins are a powerful means of physically associating enzymes in a modular fashion. By analyzing the metabolic and protein-protein interactions networks of Escherichia coli, yeast and humans, we are able to show that all three species have many more indirect protein interactions linking enzymes that share metabolites than would be expected by chance. Moreover, these interactions are distributed non-randomly in the metabolic network. Our analyses in yeast and E. coli show that reactions possessing such interactions also show higher flux than do those lacking them. On the basis of these observations, we suggest that an important role of protein interactions with mediator proteins is to contribute to the spatial organization of the cell. This hypothesis is supported by the fact that these mediator proteins are also enriched with annotations related to signal transduction, a system where scaffolding proteins are known to limit cross-talk by controlling spatial localization.     

Substrate channeling and enzyme complexes for biotechnological applications

Substrate channeling is a process of transferring the product of one enzyme to an adjacent cascade enzyme or cell without complete mixing with the bulk phase. Such phenomena can occur in vivo, in vitro, or ex vivo. Enzyme–enzyme or enzyme–cell complexes may be static or transient. In addition to enhanced reaction rates through substrate channeling in complexes, numerous potential benefits of such complexes are protection of unstable substrates, circumvention of unfavorable equilibrium and kinetics imposed, forestallment of substrate competition among different pathways, regulation of metabolic fluxes, mitigation of toxic metabolite inhibition, and so on. Here we review numerous examples of natural and synthetic complexes featuring substrate channeling. Constructing synthetic in vivo, in vitro or ex vivo complexes for substrate channeling would have great biotechnological potentials in metabolic engineering, multi-enzyme-mediated biocatalysis, and cell-free synthetic pathway biotransformation (SyPaB).     

Remarks on the supramolecular organization of the glycolytic system in vivo

The great latent catalytic capacity, manifested at the extremely high intracellular concentrations and in large apparent kcat/Km values, of the glycolytic enzymes on the one hand and their tendency in experiments in vitro to form functionally-specific flux-enhancing (channeling) complexes on the other, is considered and discussed as an apparent discrepancy. A random association of glycolytic enzymes in vivo is probable.     

Quantitative estimation of channeling from early glycolytic intermediates to CO in intact Escherichia coli

A pathway intermediate is said to be 'channeled' when an intermediate just made in a pathway has a higher probability of being a substrate for the next pathway enzyme compared with a molecule of the same species from the aqueous cytoplasm. Channeling is an important phenomenon because it might play a significant role in the regulation of metabolism. Whereas the usual mechanism proposed for channeling is the (often) transient interaction of sequential pathway enzymes, many of the supporting data come from results with pure enzymes and dilute cell extracts. Even when isotope dilution techniques have utilized whole-cell systems, most often only a qualitative assessment of channeling has been reported. Here we develop a method for making a quantitative calculation of the fraction channeled in glycolysis from in vivo isotope dilution experiments. We show that fructose-1,6-bisphosphate, in whole cells of Escherichia coli, was strongly channeled all the way to CO2, whereas fructose-6-phosphate was not. Because the signature of channeling is lost if any downstream intermediate prior to CO2 equilibrates with molecules in the aqueous cytosol, it was not possible to evaluate whether glucose-6-phosphate was channeled in its transformation to fructose-6-phosphate. The data also suggest that, in addition to pathway enzymes being associated with one another, some are free in the aqueous cytosol. How sensitive the degree of channeling is to growth or experimental conditions remains to be determined.     

Ionic strength dependence of F-actin and glycolytic enzyme associations: a Brownian dynamics simulations approach

The association of glycolytic enzymes with F-actin is proposed to be one mechanism by which these enzymes are compartmentalized, and, as a result, may possibly play important roles for: regulation of the glycolytic pathway, potential substrate channeling, and increasing glycolytic flux. Historically, in vitro experiments have shown that many enzyme/actin interactions are dependent on ionic strength. Herein, Brownian dynamics (BD) examines how ionic strength impacts the energetics of the association of F-actin with the glycolytic enzymes: lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-1,6-bisphosphate aldolase (aldolase), and triose phosphate isomerase (TPI). The BD simulations are steered by electrostatics calculated by Poisson-Boltzmann theory. The BD results confirm experimental observations that the degree of association diminishes as ionic strength increases but also suggest that these interactions are significant, at physiological ionic strengths. Furthermore, BD agrees with experiments that muscle LDH, aldolase, and GAPDH interact significantly with F-actin whereas TPI does not. BD indicates similarities in binding regions for aldolase and LDH among the different species investigated. Furthermore, the residues responsible for salt bridge formation in stable complexes persist as ionic strength increases. This suggests the importance of the residues determined for these binary complexes and specificity of the interactions. That these interactions are conserved across species, and there appears to be a general trend among the enzymes, support the importance of these enzyme-F-actin interactions in creating initial complexes critical for compartmentation.     

Molecules that initiate or help stimulate the acrosome reaction by their interaction with the mammalian sperm surface

This review deals with exogenous molecules that stimulate the acrosome reaction (AR) of mammalian sperm in vitro, presumably by acting at the sperm surface. Such molecules may exert their effect(s) by stimulating capacitation and/or by stimulation or initiation of the AR, and they are probably present at one of three putative in vivo sites (also discussed here) for the AR of a fertilizing sperm: the oviductal fluid, the cumulus oophorus matrix, and the zona pellucida. The molecules discussed include serum albumin, hydrolytic enzymes (particularly proteases); hormones including biogenic amines, estradiol, and arachidonic acid metabolites; sulfur-containing beta-amino acids; glycosaminoglycans such as hyaluronic acid; and a zona pellucida glycoprotein. Possible mechanisms to explain the effects of these molecules are also discussed. Several conclusions and suggestions are offered in this review: There is more than one site for the AR of a fertilizing sperm in vitro, depending on experimental conditions and species, but the site(s) at which the AR of a fertilizing sperm occur(s) in vivo is/are still a matter of disagreement; there are a number of molecules that can stimulate or initiate the AR in vitro, and such molecular duplication may also exist in vivo to ensure fertility; and synergistic interaction between some of those exogenous molecules may occur in the stimulation of capacitation and the stimulation or initiation of the AR.     

Implications of treadmilling for the stability and polarity of actin and tubulin polymers in vivo

In this report, we examine how the cell can selectively stabilize anchored filaments and suppress spontaneous filament assembly. Because microtubules and actin filaments have an organized distribution in cells, the cell must have a mechanism for suppressing spontaneous and random polymerization. Though the mechanism for suppressing spontaneous polymerization is unknown, an unusual property of these filaments has been demonstrated recently, i.e., under steady-stae conditions, in vitro actin filaments and microtubules can exhibit a flux of subunits through the polymers called "treadmilling." In vivo, however, most, if not all, of these polymers are attached at one end to specific structures and treadmilling should not occur. The function of treadmilling in vivo is, therefore, unclear at present. However, as shown here, the same physicochemical property of coupling assembly to ATP or GTP hydrolysis that leads to treadmilling in vitro can act to selectively stabilize anchored polymers in vivo. I show here that the theory of treadmilling implies that the concentration of subunits necessary for assembly of the nonanchored polymer will in general be higher than the concentration necessary for the assembly of polymers anchored with a specific polarity. This disparity in the monomer concentrations required for assembly can lead to a selective stabilization of anchored polymers and complete suppression of spontaneous polymerization at apparent equilibrium in vivo. It is possible, therefore, that the phenomenon of treadmilling is an in vitro manifestation of a mechanism designed to use ATP or GTP hydrolysis to control the spatial organization of filaments in the cell.     

Glycolytic enzymes associate dynamically with mitochondria in response to respiratory demand and support substrate channeling

In Arabidopsis thaliana, enzymes of glycolysis are present on the surface of mitochondria and free in the cytosol. The functional significance of this dual localization has now been established by demonstrating that the extent of mitochondrial association is dependent on respiration rate in both Arabidopsis cells and potato (Solanum tuberosum) tubers. Thus, inhibition of respiration with KCN led to a proportional decrease in the degree of association, whereas stimulation of respiration by uncoupling, tissue ageing, or overexpression of invertase led to increased mitochondrial association. In all treatments, the total activity of the glycolytic enzymes in the cell was unaltered, indicating that the existing pools of each enzyme repartitioned between the cytosol and the mitochondria. Isotope dilution experiments on isolated mitochondria, using (13)C nuclear magnetic resonance spectroscopy to monitor the impact of unlabeled glycolytic intermediates on the production of downstream intermediates derived from (13)C-labeled precursors, provided direct evidence for the occurrence of variable levels of substrate channeling. Pull-down experiments suggest that interaction with the outer mitochondrial membrane protein, VDAC, anchors glycolytic enzymes to the mitochondrial surface. It appears that glycolytic enzymes associate dynamically with mitochondria to support respiration and that substrate channeling restricts the use of intermediates by competing metabolic pathways.     

Function of Cancer Associated Genes Revealed by Modern Univariate and Multivariate Association Tests

Copy number variation (CNV) plays a role in pathogenesis of many human diseases, especially cancer. Several whole genome CNV association studies have been performed for the purpose of identifying cancer associated CNVs. Here we undertook a novel approach to whole genome CNV analysis, with the goal being identification of associations between CNV of different genes (CNV-CNV) across 60 human cancer cell lines. We hypothesize that these associations point to the roles of the associated genes in cancer, and can be indicators of their position in gene networks of cancer-driving processes. Recent studies show that gene associations are often non-linear and non-monotone. In order to obtain a more complete picture of all CNV associations, we performed omnibus univariate analysis by utilizing dCov, MIC, and HHG association tests, which are capable of detecting any type of association, including non-monotone relationships. For comparison we used Spearman and Pearson association tests, which detect only linear or monotone relationships. Application of dCov, MIC and HHG tests resulted in identification of twice as many associations compared to those found by Spearman and Pearson alone. Interestingly, most of the new associations were detected by the HHG test. Next, we utilized dCov''s and HHG''s ability to perform multivariate analysis. We tested for association between genes of unknown function and known cancer-related pathways. Our results indicate that multivariate analysis is much more effective than univariate analysis for the purpose of ascribing biological roles to genes of unknown function. We conclude that a combination of multivariate and univariate omnibus association tests can reveal significant information about gene networks of disease-driving processes. These methods can be applied to any large gene or pathway dataset, allowing more comprehensive analysis of biological processes.     

Interaction of folylpolyglutamates with enzymes in one-carbon metabolism

Of all the coenzymes, tetrahydrofolate exhibits the most structural diversity. The relationship of these structural forms to physiological function is under intense study by numerous research groups. In textbooks, tetrahydrofolate (tetrahydropteroylmonoglutamate) is shown as the coenzyme of one-carbon metabolism, but it has been known for several decades that the physiologically active forms of the coenzyme contain from 4 to 7 glutamyl residues linked by amide bonds through the gamma-carboxyl group. These glutamyl residues do not serve a direct function in transferring the one-carbon group. The tetrahydrofolylpolyglutamates were originally thought to be simply storage forms of the coenzyme, but studies now show that the polyglutamate chain of the coenzyme affects the transport properties of the coenzyme, alters the kinetic properties of many enzymes in one-carbon metabolism, and results in channeling of the coenzyme between several enzymes. In general, the dissociation constants of this group of enzymes for the tetrahydrofolylpolyglutamates are very low, in the 0.1 to 1 microM range. The concentration of the coenzyme in the cell appears to be similar to the concentration of folate-utilizing enzymes, suggesting that the concentration of unbound coenzyme in the cell may be very low. Several of the enzymes in one-carbon metabolism are either multifunctional proteins or multienzyme complexes. An active area of research is to determine if there is a functional relationship between these multifunctional enzymes and the polyglutamate portion of the coenzyme.     

In Vitro Incorporation of Molybdate into Demolybdoproteins in Escherichia coli

When Escherichia coli was grown in the presence of tungstate, inactive forms of two molybdoenzymes, nitrate reductase and formate dehydrogenase, accumulated and were converted to their active forms upon incubation of cell suspensions with molybdate and chloramphenicol. The conversion to the active enzymes did not occur in cell extracts. When incubated with [(99)Mo]molybdate and chloramphenicol, the tungstate-grown cells incorporated (99)Mo into protein components which were released from membranes by procedures used to release nitrate reductase and formate dehydrogenase and which migrated with these activities on polyacrylamide gels. Although neither activity was formed during incubation of the crude extract with molybdate, (99)Mo was incorporated into protein components which were released from the membrane fraction under the same conditions and were similar to the active enzymes in their electrophoretic properties. The in vitro incorporation of (99)Mo occurred specifically into these components and was equal to or greater than the amount incorporated in vivo under the same conditions. Molybdenum in preformed, active nitrate reductase and formate dehydrogenase did not exchange with [(99)Mo]molybdate, demonstrating that the observed incorporation depended on the demolybdo forms of the enzymes. We conclude that molybdate may be incorporated into the demolybdo forms both in vivo and in vitro; some unknown additional factor or step, required for active enzyme formation, occurs in vivo but not in vitro under the conditions employed.     

The bacterial phosphotransferase system: kinetic characterization of the glucose, mannitol, glucitol, and N-acetylglucosamine systems

The kinetic mechanisms by which the glucose, glucitol, N-acetylglucosamine, and mannitol enzymes II catalyze sugar phosphorylation have been investigated in vitro. Lineweaver-Burk analyses indicate that the glucose and glucitol enzymes II catalyze sugar phosphorylation by a sequential mechanism when the two substrates are phospho-enzyme III and sugar. The N-acetylglucosamine and mannitol enzymes II, which do not function with an enzyme III, catalyze sugar phosphorylation by a ping-pong mechanism when the two substrates are phospho-HPr and sugar. These results, as well as previously published kinetic characterizations, suggest a common kinetic mechanism for all enzymes II of the system. It is suggested that all enzymes II and enzyme II-III pairs arose from a single (fused) gene product containing two sites of phosphorylation and that phosphoryl transfer from the second phosphorylation site to sugar can only occur when the enzyme II-III pair is present in the associated state.     

Atomistic simulations of competition between substrates binding to an enzyme

Although the idea that electrostatic potentials generated by enzymes can guide substrates to active sites is well established, it is not always appreciated that the same potentials can also promote the binding of molecules other than the intended substrate, with the result that such enzymes might be sensitive to the presence of competing molecules. To provide a novel means of studying such "electrostatic competition" effects, computer simulation methodology has been developed to allow the diffusion and association of many solute molecules around a single enzyme to be simulated. To demonstrate the power of the methodology, simulations have been conducted on an artificial fusion protein of citrate synthase (CS) and malate dehydrogenase (MDH) to assess the chances of oxaloacetate being channeled between the MDH and CS active sites. The simulations demonstrate that the probability of channeling is strongly dependent on the concentration of the initial substrate (malate) in the solution. In fact, the high concentrations of malate used in experiments appear high enough to abolish any channeling of oxaloacetate. The simulations provide a resolution of a serious discrepancy between previous simulations and experiments and raise important questions relating to the observability of electrostatically mediated substrate channeling in vitro and in vivo.     

Five enzymes of the glycolytic pathway serve as substrates for purified epidermal-growth-factor-receptor kinase.

Several enzymes of the glycolytic pathway are phosphorylated in vitro and in vivo by retroviral transforming protein kinases. These substrates include the enzymes phosphoglycerate mutase (PGM), enolase and lactate dehydrogenase (LDH). Here we show that purified EGF (epidermal growth factor)-receptor kinase phosphorylates the enzymes PGM and enolase and also the key regulatory enzymes of the glycolytic pathway, phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in an EGF-dependent manner. Stoichiometry of phosphate incorporation into GAPDH (calculated from native Mr) is the highest, reaching approximately 1. LDH and other enzymes of the glycolytic pathway are not phosphorylated by the purified EGF-receptor kinase. These enzymes are phosphorylated under native conditions, and the Km values of EGF-receptor kinase for their phosphorylation are close to the physiological concentrations of these enzymes in the cell. EGF stimulates the reaction by 2-5-fold by increasing the Vmax. without affecting the Km of this process. Phosphorylation is rapid at 22 degrees C and at higher temperatures. However, unlike the self-phosphorylation of EGF-receptor, which occurs at 4 degrees C, the glycolytic enzymes are poorly phosphorylated at this temperature. Some enzymes, in particular enolase, increase the receptor Km for ATP in the autophosphorylation process and thus may act as competitive inhibitors of EGF-receptor self-phosphorylation. On the basis of the Km values of EGF receptor for the substrate enzymes and for ATP in the phosphorylation reaction, these enzymes may also be substrates in vivo for the EGF-receptor kinase.