Reciprocal influence of glucose anomers upon their respective phosphorylation by hexokinase

The phosphorylation of D-glucose (1.0mM) was measured in homogenates of tumoral islet cells incubated at 7 degrees C in the presence of labelled alpha- and/or beta-D-glucose, with or without exogenous glucose 6-phosphate. The close-to-maximal reaction velocity of hexokinase was higher with beta- than alpha-D-glucose. The latter anomer inhibited beta-D-glucose phosphorylation more than the beta-anomer decreased the phosphorylation of alpha-D-glucose. This behaviour was accounted for by the higher affinity of hexokinase for alpha- than for beta-D-glucose. These direct measurements of the relative contribution of each anomer to the overall rate of glucose phosphorylation in the presence of mixed populations of alpha- and beta-D-glucose validate the concept that the phosphorylation of D-glucose displays anomeric specificity even when the hexose is used at anomeric equilibrium. Glucose 6-phosphate inhibited the phosphorylation of the two anomers more severely when alpha-D-glucose rather than beta-D-glucose was the most abundant anomer.     

Kinetic regulation of hexokinase activity in a heterogeneous branched bienzyme system

The kinetic behaviour of a heterogeneous branched bienzyme system of beta-D-glucose oxidase and hexokinase on glucose has been studied. In this sequence, hexokinase is inhibited by its product glucose 6-phosphate and also by D-gluconic acid produced from the parallel enzymic reaction of glucose oxidase. Effect of glucose concentrations on the product's distribution in branched pathway of the bienzyme system is dependent on the kinetic properties of hexokinase and glucose oxidase. Product inhibitions, which are also pH dependent, have a strong regulatory role on the reaction flux.     

Phosphoglucomutase: its role in the response of pancreatic islets to glucose epimers and anomers

Rat pancreatic islets display phosphoglucomutase activity. The velocity of glucose-1-phosphate conversion to glucose-6-phosphate is increased in a dose-related fashion by glucose-1,6-bisphosphate. The islet homogenate, like purified muscle phosphoglucomutase, also catalyzes the synthesis of glucose-1,6-bisphosphate from glucose-6-phosphate and fructose-1,6-bisphosphate. The rate of the latter reaction is about 10,000 times lower than that of glucose-1-phosphate conversion to glucose-6-phosphate in the presence of glucose-1,6-bisphosphate. D-glucose and D-mannose, but not D-galactose nor D-fructose, markedly increase the islet content in glucose-1,6-bisphosphate. Such a content is twice higher in islets exposed for 5 minutes to alpha-D-glucose than in islets exposed to beta-D-glucose. The process of glucose-1,6-bisphosphate synthesis, as catalyzed by the alpha-stereospecific phosphoglucomutase, may play a role in the metabolic and, hence, secretory responses of the islets to glucose epimers and anomers.     

Discrimination of glucose anomers by glucokinase from liver and transplantable insulinoma

Phosphorylation of alpha- and beta-D-glucose by glucokinase from rat liver or a radiation-induced, transplantable insulinoma was investigated. Glucokinase partially purified by ion exchange chromatography on DEAE-Cibacron blue F3GA agarose was incubated for brief periods (1 or 3 min) with glucose anomers. Glucokinase from both liver and insulinoma tissue had a higher affinity for alpha-D-glucose (S0.5 = 6-7 mM) than beta-D-glucose (S0.5 = 12-14 mM). The maximum velocity was 15-20% lower for alpha-D-glucose than beta-D-glucose. Cooperative rate dependence with respect to glucose concentration was observed with both anomers (nH = 1.4). These kinetic data imply that both anomers of glucose are phosphorylated by glucokinase, however, at the physiological range of glucose concentrations below 15 mM, the higher affinity of alpha-D-glucose results in higher rates than with beta-D-glucose. At clearly pathological glucose concentrations exceeding 20 mM, the observed velocities are slightly higher with beta- than alpha-D-glucose. Glucokinase is thought to be the glucose sensor of pancreatic beta cells. The present data indicating a preferential phosphorylation of alpha-D-glucose compared to beta-D-glucose by glucokinase, supports the glucokinase-glucose sensor hypothesis, because it parallels the well established greater potency of alpha-D-glucose as a stimulant of insulin release.     

Homologs of the Rml enzymes from Salmonella enterica are responsible for dTDP-beta-L-rhamnose biosynthesis in the gram-positive thermophile Aneurinibacillus thermoaerophilus DSM 10155

The glycan chains of the surface layer (S-layer) glycoprotein from the gram-positive, thermophilic bacterium Aneurinibacillus (formerly Bacillus) thermoaerophilus strain DSM 10155 are composed of L-rhamnose- and D-glycero-D-manno-heptose-containing disaccharide repeating units which are linked to the S-layer polypeptide via core structures that have variable lengths and novel O-glycosidic linkages. In this work we investigated the enzymes involved in the biosynthesis of thymidine diphospho-L-rhamnose (dTDP-L-rhamnose) and their specific properties. Comparable to lipopolysaccharide O-antigen biosynthesis in gram-negative bacteria, dTDP-L-rhamnose is synthesized in a four-step reaction sequence from dTTP and glucose 1-phosphate by the enzymes glucose-1-phosphate thymidylyltransferase (RmlA), dTDP-D-glucose 4,6-dehydratase (RmlB), dTDP-4-dehydrorhamnose 3,5-epimerase (RmlC), and dTDP-4-dehydrorhamnose reductase (RmlD). The rhamnose biosynthesis operon from A. thermoaerophilus DSM 10155 was sequenced, and the genes were overexpressed in Escherichia coli. Compared to purified enterobacterial Rml enzymes, the enzymes from the gram-positive strain show remarkably increased thermostability, a property which is particularly interesting for high-throughput screening and enzymatic synthesis. The closely related strain A. thermoaerophilus L420-91(T) produces D-rhamnose- and 3-acetamido-3,6-dideoxy-D-galactose-containing S-layer glycan chains. Comparison of the enzyme activity patterns in A. thermoaerophilus strains DSM 10155 and L420-91(T) for L-rhamnose and D-rhamnose biosynthesis indicated that the enzymes are differentially expressed during S-layer glycan biosynthesis and that A. thermoaerophilus L420-91(T) is not able to synthesize dTDP-L-rhamnose. These findings confirm that in each strain the enzymes act specifically on S-layer glycoprotein glycan formation.     

Intramembraneous localization of rat liver microsomal hexose-6-phosphate dehydrogenase and membrane permeability to its substrates.

A method for purifying hexose-6-phosphate dehydrogenase (beta-D-glucose: NAD(P) -oxidoreductase, EC 1.1.1.47) from rat liver microsomes is described. The purified enzyme was shown to be homogeneous by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis. It is shown that the enzyme is bound to the inner surface of microsomal membranes, and that glucose 6-phosphate, but not NADP, penetrates almost freely into the membranes at 37 degrees C.     

Sucrose and starch metabolism in carrot (Daucus carota L.) cell suspensions analysed by 13-labelling: indications for a cytosol and a plastid-localized oxidative pentose phosphate pathway

Cells were grown in batch culture on a mixture of 50 mM glucose and fructose as the carbon source; either the glucose or the fructose was [1-¹³C]-labelled. In order to investigate the uptake and conversion of glucose and fructose during long-term labelling experiments in cell suspensions of Daucus carota L., samples were taken every 2 d during a 2 week culture period and sucrose and starch were assayed by means of HPLC and ¹³C-nuclear magnetic resonance. The fructose moieties of sucrose had a lower labelling percentage than the glucose moieties. Oxidative pentose phosphate pathway activity in the cytosol is suggested to be responsible for this loss of label of especially C-1 carbons. A combination of oxidative pentose phosphate pathway activity, a relatively high activity of pathway to sucrose synthesis and a slow equilibration between glucose-6-phosphate and fructose-6-phosphate could explain these results. Starch contained glucose units with a much lower labelling percentage than glucose moieties of sucrose: it was concluded that a second, plastid-localized, oxidative pentose phosphate pathway was responsible for removal of C-1 carbons of the glucosyl units used for synthesis of starch. Redistribution of label from [1-¹³C]-hexoses to [6-¹³C]-hexoses also occurred: 18-45% of the label was found at the C-6 carbons. This is a consequence of cycling between hexose phosphates and those phosphates in the cytosol catalysed by PFP. The results indicate that independent (oxidative pentose phosphate pathway mediated) sugar converting cycles exist in the cytosol and plastid.Key words: Daucus carotaL., cell suspensions, carbon-13 nuclear magnetic resonance, ¹³C-NMR, carbohydrate cycling, oxidative pentose phosphate pathway, plastid.      

The reduction of CrVI to CrIII by the alpha and beta anomers of D-glucose in dimethyl sulfoxide. A comparative kinetic and mechanistic study

The reduction of CrVI by alpha-D-glucose and beta-D-glucose was studied in dimethyl sulfoxide in the presence of pyridinium p-toluensulfonate, a medium where mutarotation is slower than the redox reaction. The two anomers reduce CrVI by formation of an intermediate CrVI ester precursor of the slow redox step. The equilibrium constant for the formation of the intermediate chromic ester and the rate of the redox steps are different for each anomer. alpha-D-Glucose forms the CrVI-Glc ester with a higher equilibrium constant than beta-D-glucose, but the electron transfer within this complex is slower than for the beta anomer. The difference is attributed to the better chelating ability of the 1,2-cis-diolate moiety of the alpha anomer. The CrV species, generated in the reaction mixture, reacts with the two anomers at a rate comparable with that of CrVI. The EPR spectra show that the alpha anomer forms several linkage isomers of the five-coordinate CrV bis-chelate, while beta-D-glucose affords a mixture of six-coordinate CrV monochelate and five-coordinate CrV bis-chelate. The conversion of the CrV mono- to bis-chelate is discussed in terms of the ability of the 1,2-cis- versus 1,2-trans-diolate moieties of the glucose anomers to bind CrV.     

Metabolism of D-glucose anomers in rat pancreatic islets exposed to equilibrated D-glucose.

This study aims at establishing the contribution of alpha- and beta-D-glucose to the total generation of (3)HOH by rat pancreatic islets exposed to D-[2 - (3)H]glucose or D-[5 - (3)H] glucose at anomeric equilibrium. The islets were incubated for 60 min at 4 degrees C in the presence of equilibrated D-glucose (2.8 and 8.3 mM) mixed with tracer amounts of either alpha- or beta-D-glucose labelled with tritium on either the C (2) or C (5) of the hexose. Relative to their respective concentrations, (3)HOH generation from the anomers labelled with tritium on the C (2) or C (5) of the hexose provided beta/alpha ratios comparable to those previously found at both 2.8 and 8.3 mM, when the islets were exposed to each anomer separately. The relative contributions of each anomer to the total generation of (3)HOH was also close to the theoretical values derived from mathematical models for the catabolism of D-glucose at anomeric equilibrium in rat islets at both 2.8 and 8.3 mM and in the case of both D-[2 - (3)H]glucose and D-[5 - (3)H]glucose. Thus, even in islets exposed to D-glucose at anomeric equilibrium, the metabolic fate of alpha-D-glucose differs vastly from that of beta-D-glucose, the enzyme-to-enzyme channelling between hexokinase isoenzymes, especially glucokinase, and phosphoglucoisomerase being restricted to alpha-D-glucose 6-phosphate.     

Characterization of vanadate-based transition-state-analogue complexes of phosphoglucomutase by spectral and NMR techniques

Near ultraviolet spectral studies were conducted on two inhibitor complexes obtained by treating the dephospho form of the phosphoglucomutase.Mg2+ complex with inorganic vanadate in the presence of either glucose 1-phosphate [cf. Percival, M. D., Doherty, K., & Gresser, M. J. (1990) Biochemistry (first of four papers in this issue)] or glucose 6-phosphate. Part of the spectral differences between the two inhibitor complexes arises because the glucose phosphate moiety in the complex derived from glucose 1-phosphate binds to the enzyme in a different way from the glucose phosphate moiety in the complex derived from glucose 6-phosphate and because these alternative binding modes produce different environmental effects on the aromatic chromophores of the dephospho enzyme. These spectral differences are strikingly similar to those induced by the binding of glucose 1-phosphate and glucose 6-phosphate to the phospho enzyme--which shows that the glucose 1-phosphate and glucose 6-phosphate moieties occupy positions in the inhibitor complexes closely related to those that they occupy in their respective catalytically competent complexes. This binding congruity indicates that in the inhibitor complexes the oxyvanadium grouping is bound at the site where (PO3-) transfer normally occurs. 31P NMR studies of the phosphate group in these complexes also provide support for this binding pattern. A number of other systems based on compounds with altered structures, such as the deoxysugar phosphates, or systems with different compositions, as in the case of the metal-free enzyme or of the glucose phosphates plus nitrate, also were examined for evidence that complexes analogous to the inhibitor complexes were formed, but none was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anomeric specificity of D-glucose metabolism in rat adipocytes

The anomeric specificity of D-glucose metabolism was investigated in rat adipocytes exposed for 60 min at 8 degrees C to pure alpha- or beta-D-glucose or to equilibrated D-glucose. The rate of D-[5-3H]glucose utilization was higher with alpha- than beta-D-glucose. However, as judged from the oxidation of D-[1-14C]glucose and D-[6-14C]glucose anomers, the fraction of D-glucose catabolism occurring via the pentose cycle was higher with beta- than alpha-D-glucose. In the presence of equilibrated D-glucose, the utilization of alpha-D-[5-3H]glucose and the oxidation of both alpha-D-[1-14C]glucose and alpha-D-[6-14C]glucose were higher, relative to the anomer concentration, than the corresponding values for beta-D-glucose. It is concluded that the anomeric specificity of D-glucose metabolism is operative in adipocytes, even when they are exposed to equilibrated D-glucose.     

Functional characterization of the initiation enzyme of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus NRS 2004/3a

The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of repeating units [-->2)-alpha-l-Rhap-(1-->3)-beta-l-Rhap-(1-->2)-alpha-l-Rhap-(1-->], with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three alpha-l-rhamnose residues, and a beta-d-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis of Geobacillus stearothermophilus NRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP from Salmonella enterica. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains of Escherichia coli and Salmonella enterica serovar Typhimurium.     

Partial purification and some properties of beta-phosphoglucomutase from Lactobacillus brevis

A phosphoglucomutase (beta-phosphoglucomutase) specific for beta-glucose 1-phosphate, which catalyzes the beta-glucose 1-phosphate:glucose 6-phosphate interconversion, was 560-fold purified from Lactobacillus brevis strain L6. The isoelectric point of beta-phosphoglucomutase was 3.8 and it had an apparent molecular weight of 29,000 estimated by gel chromatography. The enzyme required a divalent cation (Mn2+ greater than Mg2+ greater than Ni2+ greater than Co2+) and beta-glucose 1,6-bisphosphate for activity. The equilibrium constant Ke for the reaction beta-D-glucose 1-phosphate in equilibrium D-glucose 6-phosphate at 30 degrees C and pH 6.7 is 18.5. beta-phosphoglucomutase had a pH optimum between 6.3 and 6.8 and appeared to be quite specific: alpha-glucose 1-phosphate, alpha- or beta-galactose 1-phosphate and alpha- or beta-N-acetylglucosamine 1-phosphate did not substitute for beta-glucose 1-phosphate. Double reciprocal plots of the data from initial velocity studies at five beta-glucose 1-phosphate concentrations (10 to 100 microM) and four beta-glucose 1,6-bisphosphate concentrations (0.125 to 1.0 microM) showed that the apparent Michaelis constants for beta-glucose 1-phosphate and beta-glucose 1,6-bisphosphate were related to the concentrations of beta-glucose 1,6-bisphosphate and beta-glucose 1-phosphate, respectively, in such a way as to suggest a ping-pong mechanism. The same conclusion was obtained when substrate-velocity relationships were investigated at fixed ratio of both substrates: the Lineweaver-Burk plots showed linear lines and no parabolic ones. The "true" Km for beta-glucose 1-phosphate and beta-glucose 1,6-bisphosphate were found to be about 12 and 0.8 microM, respectively.     

Mechanism and contribution of the pentose phosphate cycle to glucose metabolism in epididymal fat tissue

1. Glucose 6-phosphate, fructose 6-phosphate and altroheptulose 7-phosphate are the major products formed non-oxidatively from ribose 5-phosphate by rat epididymal fat pad enzymes. 2. Arabinose 5-phosphate was detected among the reaction products and significant activity of the new enzyme of the L-type pentose pathway, D-glycero D-ido octulose 1,8-bisphosphate: D-altroheptulose 7-phosphotransferase was found. 3. The glucose moieties of glucose 1-phosphate, glucose 6-phosphate and glucose 1,6-bisphosphate were degraded and showed that epididymal fat pad enzymes relocate 14C from [2-14C]glucose into C-1, C-2, and C-3 of each hexose-phosphate. 4. The 14C-distribution patterns in the hexose-phosphates revealed that these intermediates were not in isotopic equilibrium and the rate of the transaldolase exchange reaction was relatively small. 5. The 14C-distribution data suggest that glucose 1-phosphate, rather than glucose 6-phosphate, is the first intermediate in the path of glycogen synthesis from glucose in this tissue. 6. The data provide the first proof of the mechanism of the pentose pathway in adipose tissue.     

Core glycan in the yeast multicopper ferroxidase, Fet3p: A case study of N-linked glycosylation, protein maturation, and stability

Glycosylation is essential to the maintenance of protein quality in the vesicular protein trafficking pathway in eukaryotic cells. Using the yeast multicopper oxidase, Fet3p, the hypothesis is tested that core glycosylation suppresses Fet3p nascent chain aggregation during synthesis into the endoplasmic reticulum (ER). Fet3p has 11 crystallographically mapped N‐linked core glycan units. Assembly of four of these units is specifically required for localization of Fet3p to the plasma membrane (PM). Fet3 protein lacking any one of these glycan units is found in an intracellular high‐molecular mass species resolvable by blue native gel electrophoresis. Individually, the remaining glycan moieties are not required for ER exit; however, serial deletion of these by N → A substitution correlates with these desglycan species failure to exit the ER. Desglycan Fet3 proteins that localize to the PM are wild type in function indicating that the missing carbohydrate is not required for native structure and biologic activity. This native function includes the interaction with the iron permease, Ftr1p, and wild type high‐affinity iron uptake activity. The four essential sequons are found within relatively nonpolar regions located in surface recesses and are strongly conserved among fungal Fet3 proteins. The remaining N‐linked sites are found in more surface exposed, less nonpolar environments, and their conservation is weak or absent. The data indicate that in Fet3p the N‐linked glycan has little effect on the enzyme's molecular activity but is critical to its cellular activity by maximizing the protein's exit from the ER and assembly into a functional iron uptake complex.     

Identification of the defect in lipophosphoglycan biosynthesis in a non-pathogenic strain of Leishmania major.

The major macromolecule on the surface of the protozoan parasite, Leishmania major, is a complex lipophosphoglycan (LPG), which is anchored to the plasma membrane by an inositol-containing phospholipid. A defect in LPG biosynthesis is thought to be responsible for the avirulence of the L. major strain LRC L119 in mice. In order to identify the nature of this defect we have characterized two truncated forms of LPG, which are accumulated in this strain, by one- and two-dimensional 500-MHz 1H NMR spectroscopy, two-dimensional heteronuclear 1H-31P NMR spectroscopy, methylation analysis, and exoglycosidase digestions. The structures of these glycoinositolphospholipids, termed GIPL-4 and -6, are as follows: [formula: see text] The glycan moieties of GIPL-4 and -6 are identical to the anchor region of LPG, which is also substituted with a Glc-1-PO4 residue in approximately 60% of the structures. However, instead of being capped with chains of phosphorylated oligosaccharide repeat units, both glycan moieties terminate in Man alpha 1-PO4, suggesting that the defect in LPG biosynthesis is in the transfer of galactose to this residue to form the disaccharide backbone of the first repeat unit. These results indicate that the phosphoglycan moiety of LPG is essential for intracellular survival of the parasite and have implications for LPG biosynthesis.     

The relative contribution of mannose salvage pathways to glycosylation in PMI-deficient mouse embryonic fibroblast cells

Mannose for mammalian glycan biosynthesis can be imported directly from the medium, derived from glucose or salvaged from endogenous or external glycans. All pathways must generate mannose 6-phosphate, the activated form of mannose. Imported or salvaged mannose is directly phosphorylated by hexokinase, whereas fructose 6-phosphate from glucose is converted to mannose 6-phosphate by phosphomannose isomerase (PMI). Normally, PMI provides the majority of mannose for glycan synthesis. To assess the contribution of PMI-independent pathways, we used PMI-null fibroblasts to study N-glycosylation of DNase I, a highly sensitive indicator protein. In PMI-null cells, imported mannose and salvaged mannose make a significant contribution to N-glycosylation. When these cells were grown in mannose-free medium along with the mannosidase inhibitor, swainsonine, to block the salvage pathways, N-glycosylation of DNase I was almost completely eliminated. Adding approximately 13 microm mannose to the medium completely restored normal glycosylation. Treatment with bafilomycin A(1), an inhibitor of lysosomal acidification, also markedly reduced N-glycosylation of DNase I, but in this case only 8 microm mannose was required to restore full glycosylation, indicating that a nonlysosomal source of mannose made a significant contribution. Glycosylation levels were greatly also reduced in glycoconjugate-free medium, when endosomal membrane trafficking was blocked by expression of a mutant SKD1. From these data, we conclude that PMI-null cells can salvage mannose from both endogenous and external glycoconjugates via lysosomal and nonlysosomal degradation pathways.     

The preparation of an immunoglobulin--amyloglucosidase conjugate and its quantitation by an enzyme-cycling assay

The production and characterization of covalent amyloglucosidase-antibody conjugates using anti-human serum albumin immunoglobulin G are described. The conjugation procedure is based on the periodate oxidation of carbohydrate moieties that are covalently linked to the enzyme, followed by Schiff's base formation with amino residues on IgG. An ultrasensitive enzyme cycling assay for glucose, the product of maltose cleavage by amyloglucosidase, was developed in order to increase the sensitivity of detecting the enzyme-antibody conjugate. The cycling assay, which allows the accurate measurement of glucose in the picomole range, involves an enzymatic conversion of glucose to glucose-6-phosphate and then isomerization to fructose-6-phosphate. A futile cycle between fructose-6-phosphate and fructose-1,6-diphosphate results in accumulation of adenosine diphosphate at a rate proportional to the original glucose concentration. The rate was monitored by a spectrophotometric system involving pyruvate kinase, phospho(enol)pyruvate, lactate dehydrogenase, and diphosphopyridine nucleotide.     

Anomer specificity of glucose-6-phosphatase and glucokinase

The anomeric form of glucose produced by glucose-6-phosphatase was studied using an apparatus that specifically measures beta-D-glucose. The time course of beta-D-glucose formation from glucose-6-P by glucose-6-phosphatase is essentially linear. In the presence of mutarotase, this rate is reduced to 70% of that obtained in the absence of mutarotase. When detergent treated microsomes were used, the rate of beta-D-glucose formation is unaffected by mutarotase. These results suggest that only beta-anomer of glucose is produced by microsomal glucose-6-phosphatase and this specificity is determined by translocase for glucose-6-P or glucose. It was also demonstrated that alpha-D-glucose is the substrate for glucokinase.     

Genetic studies with a phosphoglucose isomerase mutant of Saccharomyces cerevisiae.

Summary A mutation pgi1 in the yeast Saccharomyces cerevisiae conferring deficiency of the glycolytic enzyme glucose 6-phosphate isomerase is characterised genetically. The mutation segregates 2⁺:2^- in tetrads from diploids heterozygous for the mutant phenotype. The mutation is semi-dominant and is located on the right arm of chromosome II in the order: tsm134-lys2-pgi1-tyr1 approximately 15 map units from tyr1. The mutation pgi1 defines the structural gene of glucose 6-phosphate isomerase and can be suppressed intragenically giving revertants that have an unstable enzyme. In one temperature-sensitive revertant no enzyme activity in excess of the mutant level could be detected although fructose 6-phosphate was converted to glucose 6-phosphate in vivo. The suppressor locus in this revertant is dominant and is unlinked to the pgi1 locus.