Yeast hexokinase 2 is known to be a phosphoprotein in vivo, prominently labeled from 32P-inorganic phosphate after a shift of cells to medium with low glucose concentration [Vojtek, A. B., & Fraenkel D. G. (1990) Eur. J. Biochem, 190, 371-375]. The principal and perhaps sole site of phosphorylation is now identified as residue serine-15, by observation of a single tryptic peptide difference, its sequencing and size determination by mass spectrometry, and by mutation to alanine, which prevents phosphorylation in vivo. Although protein kinase A was unlikely to accomplish the phosphorylation in vivo, serine-15 does belong to a protein kinase A consensus phosphorylation sequence, and in vitro phosphorylation by protein kinase A at serine-15 could be shown by labeling and by peptide determination. The alanine-15 mutant enzyme was not phosphorylated in vitro.

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Glucose phosphorylation capacity is known to be in excess of glucose flux in Saccharomyces cerevisiae wild type but not in a mutant strain lacking the two hexokinases but still having glucokinase. Nonetheless, we show here that in the latter strain, as in the wild type, the internal concentration of glucose is apparently low during growth on glucose and that additional glucokinase activity does not increase glucose flux. The glucokinasedependent strain accumulates substantial amounts of glucose internally in batch culture after exhaustion of glucose, as well as from maltose. In both of these situations, low concentrations of radioactive glucose provided to the medium are used with incomplete, if any, mixing with the internal pool. Furthermore, in contrast to activity of hexokinase and other enzymes, little glucokinase activity is revealed by toluene treatment of cells.These results may point to a connection between glucose entry and its phosphorylation by glucokinase, but separate explanations for the various findings are also possible.Saccharomyces cerevisiae has three glucose-phosphorylating enzymes: hexokinases 1 and 2 (which also phosphorylate fructose) and glucokinase (which does not); the three genes are HXK1, HXK2, and GLK1, respectively. Studies of mutants (e.g., reference 14) show that any single one of the three enzymes is adequate for growth on glucose. However, in the wild-type strain their individual roles are unclear, with hexokinase 2 predominating on glucose, hexokinase 1 being glucose repressible, and glucokinase showing lower levels of activity. As reported for a congenic series of null mutants (28), in the wild-type strain the maximum glucose-phosphorylating capacity, measured in vitro, was more than twice the glucose flux during growth, while in a strain with only glucokinase, Vma and flux values were similar and low.Those results suggest that while the kinases are normally in excess, in a strain with glucokinase alone it might limit the rate of glucose metabolism.The present study is on the physiology of strains having only glucokinase. Certain of the findings do not fit with glucokinase activity directly limiting glucose utilization in such strains but point to linkage of uptake and phosphorylation. Acid-soluble metabolite pools were, except as noted, prepared by filtration, freezing, and perchloric acid extraction (22), generally using 420 A580 units of cells (e.g., 420 ml of a culture at an A580 of 1) and Millipore RA120 filters. The frozen filter was extracted with 1 ml of 0.3 M perchloric acid containing 1 mM K2EDTA, neutralized with 0.1 volume of 3 M KHCO3, and centrifuged to remove perchlorate and debris. The cells were not washed on the filter. Determination of the volume of the trapped medium employed a variety of impermeable solutes ([14C]inulin, arabinose, gluconate, and alpha-ketoglutarate), while for determining total aqueous volume (internal plus trapped) 3H20 was used. For reasons of economy, these calibrations employed 1-ml volumes of washed cells at a high density (420A580 u...

We present a comparative study of Escherichia coli with normal and increased amounts of fructose-1,6-bisphosphate aldolase. Most experiments employed a resting cell system involving a high cell density (so as to obtain the soluble pool by direct extraction) and anaerobic incubation in the presence of chloramphenicol. Glucose use is linear with time with a rate ca. half of that in growth, fermentation is almost quantitative, and metabolite concentrations reach a quasi steady state. Increased amount of aldolase had little effect on glucose flux; fructose-1,6-P2 concentration decreased by ca. one-third, and the extent of equilibration of its two halves, measured by a dismutation procedure on samples taken during metabolism of [6-14C]glucose, increased from 0.33 [(cpm in C1-3)/(cpm in C1-6)] to 0.43. Using the simplest model, that increased amount of aldolase does not perturb net flux or later metabolites, together with the steady-state rate equations for aldolase and triose-P isomerase, we show that the results with resting cells fit with the extra enzyme being fully active, and do not necessitate special assumptions concerning a glycolytic complex, metabolite compartmentation, or secondary mechanisms assuring high metabolite concentration. However, the fit does require that the measured Vmax values substantially underestimate the actual ones. Calculation also shows that the forms of the predicted curves--and hence the fit with experimental data--of fructose-1,6-P2 concentration and labeling as a function of the amount of aldolase are highly dependent on glyceraldehyde-3-P concentration but independent of the kinetic parameters of aldolase.

Mutants have been isolated in S. cereuisiae with the phenotype of growth on pyruvate but not on glucose, or growth on rich medium with pyruvate but inhibition by glucose. Screening of mutagenized cultures was either without an enrichment step, or after enrichment using the antibiotic netropsin (YOUNG et al. 1976) or inositol starvation (HENRY, DONAHUEand CULBERTSON 1975). One class of mutants lacked pyruvate kinase (p y k), another class had all the enzymes of glycolysis, and one mutant lacked phosphoglucose isomerase (pgi, MAITRA 1971). Partial reversion of pyruvate kinase mutants on rich medium containing glucose gave double mutants now'also lacking hexokinase (hzk), phosphofructokinase (pfk), or several enzymes of glycolysis (gcr) . In diploids the mutations were recessive. pyk, pgi, pfk, and gcr segregated 2:2 from their wild-type alleles. PYK hxk, PYK pfk, and PYK gcr segregants grew on glucose.