Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and PII

Genetic and biochemical analyses showed that hexokinase PII is mainly responsible for glucose repression in Saccharomyces cerevisiae, indicating a regulatory domain mediating glucose repression. Hexokinase PI/PII hybrids were constructed to identify the supposed regulatory domain and the repression behavior was observed in the respective transformants. The hybrid constructs allowed the identification of a domain (amino acid residues 102-246) associated with the fructose/glucose phosphorylation ratio. This ratio is characteristic of each isoenzyme, therefore this domain probably corresponds to the catalytic domain of hexokinases PI and PII. Glucose repression was associated with the C-terminal part of hexokinase PII, but only these constructs had high catalytic activity whereas opposite constructs were less active. Reduction of hexokinase PII activity by promoter deletion was inversely followed by a decrease in the glucose repression of invertase and maltase. These results did not support the hypothesis that a specific regulatory domain of hexokinase PII exists which is independent of the hexokinase PII catalytic domain. Gene disruptions of hexokinases further decreased repression when hexokinase PI was removed in addition to hexokinase PII. This proved that hexokinase PI also has some function in glucose repression. Stable hexokinase PI overproducers were nearly as effective for glucose repression as hexokinase PII. This showed that hexokinase PI is also capable of mediating glucose repression. All these results demonstrated that catalytically active hexokinases are indispensable for glucose repression. To rule out any further glycolytic reactions necessary for glucose repression, phosphoglucoisomerase activity was gradually reduced. Cells with residual phosphoglucoisomerase activities of less than 10% showed reduced growth on glucose. Even 1% residual activity was sufficient for normal glucose repression, which proved that additional glycolytic reactions are not necessary for glucose repression. To verify the role of hexokinases in glucose repression, the third glucose-phosphorylating enzyme, glucokinase, was stably overexpressed in a hexokinase PI/PII double-null mutant. No strong effect on glucose repression was observed, even in strains with 2.6 U/mg glucose-phosphorylating activity, which is threefold increased compared to wild-type cells. This result indicated that glucose repression is only associated with the activity of hexokinases PI and PII and not with that of glucokinase.     

Xylose induced decrease in hexokinase PII activity confers resistance to carbon catabolite repression of invertase synthesis in Saccharomyces carlsbergensis

The relationship between the xylose induced decrease in hexokinase PII activity and the derepression of invertase synthesis in yeast is described. When xylose was added to cells growing in a chemostat under nitrogen limitation, the catabolic repression was supressed as shown by the large increase on invertase levels even if glucose remained high. The glucose phosphorylating-enzymes were separated by hydroxylapatite chromatography and it is shown that the treatment with xylose is accompanied by a loss of 98% hexokinase PII and a 50% of the PI isoenzyme, whereas the levels of glucokinase as well as those of glucose-6-phosphate, fructose-6-phosphate, pyruvate and ATP remained unaffected.The analysis of the enzymes present in cells grown in ethanol, limiting glucose and high glucose, shows that hexokinase PII predominates in cells under catabolic repression, the opposite is true for glucokinase, whereas hexokinase PI remains unaffected.     

31P NMR and 13C NMR studies of recombinant Saccharomyces cerevisiae with altered glucose phosphorylation activities

Manipulation of cellular metabolism to maximize the yield and rate of formation of desired products may be achieved through genetic modification. Batch fermentations utilizing glucose as a carbon source were performed for three recombinant strains of Saccharomyces cerevisiae in which the glucose phosphorylation step was altered by mutation and genetic engineering. The host strain (hxk1 hxk2 glk) is unable to grow on glucose or fructose; the three plasmids investigated expressed hexokinase PI, hexokinase PII, or glucokinase, respectively, enabling more rapid glucose and fructose phosphorylation in vivo than that provided by wild-type yeast.Intracellular metabolic state variables were determined by ³¹P NMR measurements of in vivo fermentations under nongrowth conditions for high cell density suspensions. Glucose consumption, ethanol and glycerol production, and polysaccharide formation were determined by ¹³C NMR measurements under the same experimental conditions as used in the ³¹P NMR measurements. The trends observed in ethanol yields for the strains under growth conditions were mimicked in the nongrowth NMR conditions.Only the strain with hexokinase PI had higher rates of glucose consumption and ethanol production in comparison to healthy diploid strains in the literature. The hexokinase PII strain drastically underutilized its glucose-phosphorylating capacity. A regulation difference in the use of magnesium-free ATP for this strain could be a possible explanation. Differences in ATP levels and cytoplasmic pH values among the strains were observed that could not have been foreseen. However, cytoplasmic pH values do not account for the differences observed among in vivo and in vitro glucose phosphorylation activities of the three recombinant strains.     

Sugar repression in the methylotrophic yeast Hansenula polymorpha studied by using hexokinase-negative, glucokinase-negative and double kinase-negative mutants

Two glucose-phosphorylating enzymes, a hexokinase phosphorylating both glucose and fructose, and a glucose-specific glucokinase were electrophoretically separated in the methylotrophic yeastHansenula polymorpha. Hexokinase-negative mutants were isolated inH. polymorpha by using mutagenesis, selection and genetic crosses. Regulation of synthesis of the sugar-repressed alcohol oxidase, catalase and maltase was studied in different hexose kinase mutants. In the wild type and in mutants possessing either hexokinase or glucokinase, glucose repressed the synthesis of maltase, alcohol oxidase and catalase. Glucose repression of alcohol oxidase and catalase was abolished in mutants lacking both glucose-phosphorylating enzymes (i.e. in double kinase-negative mutants). Thus, glucose repression inH. polymorpha cells requires a glucose-phosphorylating enzyme, either hexokinase or glucokinase. The presence of fructose-phosphorylating hexokinase in the cell was specifically needed for fructose repression of alcohol oxidase, catalase and maltase. Hence, glucose or fructose has to be phosphorylated in order to cause repression of the synthesis of these enzymes inH. polymorpha suggesting that sugar repression in this yeast therefore relies on the catalytic activity of hexose kinases.     

Distribution of hexokinase isoenzymes depending on a carbon source in Saccharomyces cerevisiae.

DEAE cellulose chromatography and agar gel electrophoresis of glucose-phosphorylating enzymes in Saccharomyces cerevisiae showed the existence of glucokinase and two hexokinase isoenzymes ( designated as hexokinase I and II ). The distribution of hexokinase isoenzymes was dependent on a carbon source in the medium, while that of glucokinase was not dependent. The cells grown on 3 % ethanol as carbon source showed the isoenzyme pattern with predominant hexokinase I and a little hexokinase II. The isoenzyme pattern of the cells grown on 6 % glucose, which was differnt from that of the cells grown on ethanol, showed that hexokinase I and II were minor and major parts respectively. When the cells grown on 3 % ethanol were incubated on the medium containing 6 % glucose, hexokinase I was repressed and hexokinase II inducted. These facts suggest that two hexokinase isoenzymes, but not glucokinase, are adaptive enzyme.     

Physiological role of glucose-phosphorylating enzymes in Saccharomyces cerevisiae.

Starting with a mutant of Saccharomyces cerevisiae lacking glucokinase and both the hexokinase isozymes P1 and P2, strains were constructed, by genetic crosses, that carry single glucose-phosphorylating enzymes. The P1 and P2 isozymes and a structurally altered form of P1 hexokinase were partially purified from these strains. Hexokinases P1, P2, and the altered P1 enzyme, respectively, phosphorylate fructose nearly four, two, and ten times as fast as they phosphorylate glucose. Strains bearing P1 show a pronounced Pasteur reaction and phosphorylate glucose, fructose, and mannose faster than those bearing the P2 isozyme. However, there is no appreciable difference between these two hexokinases in regard to the rate and the extent of growth that they sustain. The ability of yeast to grow on a particular sugar is contingent only upon the presence of an enzyme that phosphorylates it. Glucokinase seems to be responsible for catalyzing nearly half of the glucose flux in the wild type yeast. Strains bearing glucokinase alone do show a Pasteur effect.     

Autophosphorylation of yeast hexokinase PII

Autophosphorylation of hexokinase PII was studied using an enzyme purified from Saccharomyces cerevisiae. Incubation of this enzyme preparation with [gamma-32P]ATP and Mn2+ or Mg2+ gave a phosphoprotein of molecular mass 58,000 which corresponded to hexokinase PII. D-Xylose stimulated autophosphorylation of hexokinase PII. Dilution of hexokinase PII over a 10-fold concentration range did not change the specific activity of hexokinase PII autophosphorylation suggesting that it may occur by an intramolecular mechanism.     

Author index to volume 140

The effects of varied durations of food deprivation on the rates and kinetics of glucose phosphorylation by isolated rat hepatocytes have been examined. Glucokinase activity was measured concurrently in extracts from these cells prepared from livers of rats which had fasted for 0, 24, 48 and 72 h. Significant levels of hepatocyte glucose phosphorylation were noted even when glucokinase levels were extrapolated to zero. The K_0.5-glucose value of 33 mM in cells from fed rats increased to 48 mM after a 72-h fast. It is concluded that a high K_0.5 glucose-phosphorylating enzyme or enzymes compensatory to insulin-dependent glucokinase is/are involved in rat liver glucose phosphorylation.     

Influence of the oligomeric state of yeast hexokinase isozymes on inactivation and unfolding by urea.

The effect of the association-dissociation equilibrium on the urea-induced inactivation and unfolding of the yeast hexokinase isoforms, PI and PII, showed that these enzymes are more stable as dimers. For the monomeric PII, the inactivation and unfolding processes occurred in parallel. However, inactivation precedes the unfolding of monomeric PI or dimeric PI and PII. The unfolding transitions are biphasic for PI indicating stable intermediates, whereas for the PII isoform the unfolding occurs in a single step. Our data suggests that although PI and PII present a 78% identity in their amino acid sequences, they probably have distinct inactivation and unfolding by urea behavior.     

Cloning of hexokinase isoenzyme PI from Saccharomyces cerevisiae: PI transformants confirm the unique role of hexokinase isoenzyme PII for glucose repression in yeasts

Summary Hexokinase isoenzyme PI was cloned using a gene pool obtained from a yeast strain having only one functional hexokinase, isoenzyme PI. The gene was characterized using 20 restriction enzymes and located within a region of 2.0 kbp. The PI plasmid strongly hybridized with the PII plasmids isolated previously (Fröhlich et al. 1984). Hence there was a close relationship between the two genes, one of which must have been derived from the other by gene duplication. In conrrast, glucose repression was restored only in hexokinase PII transformants; PI transformants remained non-repressible. This observation provided additional evidence for the hypothesis of Entian (1980) that only hexokinase PII is necessary for glucose repression. Furthermore, glucose phosphorylating activity in PI transformants exceeded that of wild-type cells, giving clear evidence that the phosphorylating capacity is not important for glucose repression.     

The progressive effects of fasting on glucose phosphorylation by isolated rat hepatocytes: the involvement of a high K0.5 enzyme

The effects of varied durations of food deprivation on the rates and kinetics of glucose phosphorylation by isolated rat hepatocytes have been examined. Glucokinase activity was measured concurrently in extracts from these cells prepared from livers of rats which had fasted for 0, 24, 48 and 72 h. Significant levels of hepatocyte glucose phosphorylation were noted even when glucokinase levels were extrapolated to zero. The K_0.5-glucose value of 33 mM in cells from fed rats increased to 48 mM after a 72-h fast. It is concluded that a high K_0.5 glucose-phosphorylating enzyme or enzymes compensatory to insulin-dependent glucokinase is/are involved in rat liver glucose phosphorylation.     

The high resolution crystal structure of yeast hexokinase PII with the correct primary sequence provides new insights into its mechanism of action

Hexokinase is the first enzyme in the glycolytic pathway, catalyzing the transfer of a phosphoryl group from ATP to glucose to form glucose 6-phosphate and ADP. Two yeast hexokinase isozymes are known, namely PI and PII. The crystal structure of yeast hexokinase PII from Saccharomyces cerevisiae without substrate or competitive inhibitor is determined and refined in a tetragonal crystal form at 2.2-A resolution. The folding of the peptide chain is very similar to that of Schistosoma mansoni and previous yeast hexokinase models despite only 30% sequence identity between them. Distinct differences in conformation are found that account for the absence of glucose in the binding site. Comparison of the current model with S. mansoni and yeast hexokinase PI structures both complexed with glucose shows in atomic detail the rigid body domain closure and specific loop movements as glucose binds. A hydrophobic channel formed by strictly conserved hydrophobic residues in the small domain of the hexokinase is identified. The channel's mouth is close to the active site and passes through the small domain to its surface. The possible role of the observed channel in proton transfer is discussed.     

Cloning of genes that complement yeast hexokinase and glucokinase mutants.

Genes complementing the glucose-negative fructose-negative Saccharomyces cerevisiae triple mutant strain (hxkl hxk2 glk1), which lacks hexokinase PI, hexokinase PII, and glucokinase, were obtained from a pool of yeast DNA in the multicopy plasmid YEp13.     

Aluminum detoxification mechanism in Pseudomonas fluorescens is dependent on iron

Abstract Hexose phosphorylation was studied in Aspergillus nidulans wild-type and in a fructose non-utilising mutant ( frA ). The data indicate the presence of at least one hexokinase and one glucokinase in wild-type A. nidulans , while the fr A1 mutant lacks hexokinase activity. The A. nidulans gene encoding hexokinase was isolated by complementation of the fr A1 mutation. The absence of hexokinase activity in the fr A1 mutant did not interfere with glucose repression of the enzymes involved in alcohol and l-arabinose catabolism. This suggests that, unlike the situation in yeast where mutation of hexokinase PII abolishes glucose repression, the A. nidulans hexokinase might not be involved in glucose repression.     

Reduced enzymatic activity of glucokinase after affinity labeling: results from spectrophotometry and electrospray ionization mass spectrometry

Glucokinase catalyzes phosphoryl group transfer from ATP to glucose to form glucose-6-phosphate in the first step of cellular metabolism. While the location of the ATP-binding site of glucokinase was proposed recently, limited information exists on its conformation or the key amino acids involved in substrate binding. Affinity labeling with phenylglyoxal is used to probe possible Arg residues involved in ATP binding. Electrospray ionization mass spectrometry indicates that reaction of purified glucokinase with phenylglyoxal results in as many as six or seven sites of modification, suggesting nonspecific modification. However, preincubation of glucokinase with glucose followed by reaction with phenylglyoxal reveals only two sites of modification. Glucokinase activity assays show that enzyme preincubated with glucose possesses residual activity corresponding to the fraction of unmodified enzyme observed by mass spectrometry, strongly suggesting that glucokinase preincubated with glucose is specifically labeled and inactivated upon modification by phenylglyoxal. The data support the existing conformational model of glucokinase.     

Identification and characterization of a novel glucose-phosphorylating enzyme in Kluyveromyces lactis

Recent data suggest that hexokinase KlHxk1 (Rag5) represents the only glucose-phosphorylating enzyme of Kluyveromyces lactis, which also is required for glucose signalling. Long-term growth studies of a K. lactis rag5 mutant, however, reveal slow growth on glucose, but no growth on fructose. Isolation of the permissive glucose-phosphorylating enzyme, mass spectrometric tryptic peptide analysis and determination of basic kinetic data identify a novel glucokinase (KlGlk1) encoded by ORF KLLA0C01,155g. In accordance with the growth characteristics of the rag5 mutant, KlGlk1 phosphorylates glucose, but fails to act on fructose as a sugar substrate. Multiple sequence alignment indicates the presence of at least one glucokinase gene in all sequenced yeast genomes.     

Genetic and biochemical evidence for hexokinase PII as a key enzyme involved in carbon catabolite repression in yeast

Summary Mutants with reduced hexokinase activity previously isolated as resistant to carbon catabolite repression of invertase and maltase (Zimmermann and Scheel, 1977) were allele tested with mutant strains of Lobo and Maitra (1977) which had defects in one or several of the genes coding for glucokinase and the two unspecific hexokinases. It could be demonstrated, that the mutation abolishing carbon catabolite repression had occurred in a gene allelic to the structural gene of hexokinase PII. Moreover, the defective mutant allele for hexokinase PII isolated by Lobo and Maitra (1977) was also defective in carbon catabolite repression. Neither glucokinase nor hexokinase PI showed any effect on this regulatory system. Biochemical analysis in crude extracts also showed altered kinetic properties of hexokinases in the hex1 mutants. The results directly support the hypothesis previously put forward, that one of the hexokinases is not only active as a catalytic, but also as a regulatory protein.     

Hexose kinases of avocado

The subcellular location and properties of enzymes concerned with the phosphorylation of glucose and fructose in avocado (Persea americana Mill. cv. Hass) have been studied. A substantial amount of glucose-phosphorylating activity was particulate and fractionation of extracts by sucrose density gradient centrifugation indicated that most of this activity was associated with the mitochondria. Three hexose-phosphorylating enzymes were resolved by DEAE-cellulose chromatography of the cytosolic fraction. These were a hexokinase (EC 2.7.1.1), which had strong preference for glucose as substrate, and two specific fructokinases (EC 2.7.1.4). ATP was the preferred phosphoryl donor for the hexose kinases of avocado.     

Glucose 6-phosphate plays a central role in the activation of glycogen synthase by glucose in hepatocytes

The state of activation of glycogen synthase enhanced by glucose, other sugars and gluconeogenic precursors shows a strong positive correlation with the intracellular concentrations of glucose 6-P when ATP concentrations remain constant. The concentrations of glucose 6-P achieved upon incubation of hepatocytes with glucose plus mannoheptulose, an inhibitor of glucokinase and hexokinase, were lower than those found when the incubation was carried out with glucose alone. Under these conditions, in keeping with the decrease in glucose 6-P, the activation of glycogen synthase by glucose was also impaired. On the other hand the inactivation of glycogen phosphorylase was not altered in the presence of mannoheptulose.     

Resistance to 2-deoxyglucose in yeast: A direct selection of mutants lacking glucose-phosphorylating enzymes

Summary When strains of Saccharomyces cerevisiae carrying a single glucose-phosphorylating enzyme such as hexokinase P1 or hexokinase P2 or glucokinase, are subjected to the selection pressure against the toxic sugar 2-deoxyglucose, the majority of survivors are mutants lacking the respective enzymes. All the 2-deoxyglucose-resistant segregants recovered from backcrosses of these mutants to a wild type strain are glucose-negative and all the sensitive ones are glucose-positive. The hexokinase mutations are located in the same complementation groups as defined by the structural genes of hexokinase P1 and hexokinase P2. No interallelic complementation has been observed either in hexokinase P1 or in hexokinase P2 amongst a total of 4×64, and 5×60 different combinations of independent mutants at the hxk1 and hxk2 loci respectively. There appears to be neither a common genetic regulator controlling two or more of these glucose-phosphorylating enzymes nor a sugar carrier that can be dispensed with.