This paper examines whether the in vivo behavior of yeast glycolysis can be understood in terms of the in vitro kinetic properties of the constituent enzymes. In nongrowing, anaerobic, compressed Saccharomyces cerevisiae the values of the kinetic parameters of most glycolytic enzymes were determined. For the other enzymes appropriate literature values were collected. By inserting these values into a kinetic model for glycolysis, fluxes and metabolites were calculated. Under the same conditions fluxes and metabolite levels were measured.In our first model, branch reactions were ignored. This model failed to reach the stable steady state that was observed in the experimental flux measurements. Introduction of branches towards trehalose, glycogen, glycerol and succinate did allow such a steady state. The predictions of this branched model were compared with the empirical behavior. Half of the enzymes matched their predicted flux in vivo within a factor of 2. For the other enzymes it was calculated what deviation between in vivo and in vitro kinetic characteristics could explain the discrepancy between in vitro rate and in vivo flux.

A large proportion of the 6,000 genes present in the genome of Saccharomyces cerevisiae, and of those sequenced in other organisms, encode proteins of unknown function. Many of these genes are "silent, " that is, they show no overt phenotype, in terms of growth rate or other fluxes, when they are deleted from the genome. We demonstrate how the intracellular concentrations of metabolites can reveal phenotypes for proteins active in metabolic regulation. Quantification of the change of several metabolite concentrations relative to the concentration change of one selected metabolite can reveal the site of action, in the metabolic network, of a silent gene. In the same way, comprehensive analyses of metabolite concentrations in mutants, providing "metabolic snapshots," can reveal functions when snapshots from strains deleted for unstudied genes are compared to those deleted for known genes. This approach to functional analysis, using comparative metabolomics, we call FANCY-an abbreviation for functional analysis by co-responses in yeast.

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By using a modified technique to measure glucose uptake in Saccharomyces cerevisiae, potential uncertainties have been identified in previous determinations. These previous determinations had led to the proposal that S. cerevisiae contained a constitutive low-affinity glucose transporter and a glucose-repressible high-affinity transporter. We show that, upon transition from glucose-repressed to -derepressed conditions, the maximum rate of glucose transport is constant and only the affinity for glucose changes. We conclude that the transporter or group of transporters is constitutive and that regulation of glucose transport occurs via a factor that modifies the affinity of the transporters and not via the synthesis of different kinetically independent transporters. Such a mechanism could, for instance, be accommodated by the binding of kinases causing a change in affinity for glucose.Until very recently, glucose uptake in Saccharomyces cerevisiae was thought to be mediated by two kinetically distinct mechanisms. A constitutive low-affinity transport system, consisting of a nonconcentrative facilitated diffusion process with a Km of approximately 20 mM (50 mM for fructose), and a kinase-dependent, glucose-repressible high-affinity transport system, also consisting of a facilitated diffusion process with a Km of approximately 1 mM (5 mM for fructose), have been described (3). The diagnostic technique used in the elucidation of these two systems was the biphasic nature of Eadie-Hofstee plots obtained from short-time-scale (5-s) uptake experiments using radiolabelled glucose. Analysis of such plots, however, is complex and requires computer-assisted nonlinear regression. Simple linearization of the two slopes in the biphasic plot can produce poor estimations of the kinetic parameters or, in severe cases, fail to detect all of the systems present (24, 26).The widespread use of these experimental and analytical techniques has led to the description of high-and low-affinity glucose transporters in several Saccharomyces species (7) as well as other yeasts such as Kluyveromyces lactis (23), Kluyveromyces marxianus (9), and Candida wickerhamii (20).More recent genetic studies have identified a number of genes involved in glucose transport in S. cerevisiae. SNF3 (sucrose nonfermenting), a glucose-repressible component of high-affinity glucose transport (4) and HXT1 and HXT2 (hexose transport) are proposed components of high-affinity glucose transport (14,16). Further studies have revealed that HXT3, HXT4, and an unidentified component are likewise involved in high-affinity glucose transport while SNF3 may be involved only in the regulation of high-affinity transport (13). While at least six components have been identified as being involved in the high-affinity uptake, the nature of the lowaffinity component has remained elusive, and this has led to questions regarding the nature of this component. Several groups have suggested that low-affinity uptake may be a consequence of passive diffusion (8,10,11 Strains and growth con...