Spectrophotometric studies on the interaction between triose phosphate isomerase and inhibitors.

The binding of ligands to chicken muscle triose phosphate isomerase was studied. Changes in u.v. absorbance of the enzyme were used to measure binding, and the dissociation constant was determined over a range of pH values. The ligands were 2-phosphoglycollate and rac-glycerol 3-phosphate (only the D-isomer, sn-glycerol 1-phosphate, binds appreciably). Non-linear regression was used to fit calculated curves to the experimental points and hence to compare different models. Both active sites in the dimeric enzyme probably bound 2-phosphoglycollate, without any interaction between the sites. The results of crystallographic analysis [phillips, Rivers, Sternberg, Thornton & Wilson (1977) Biochem. Soc Trans. 5, 642--647], and experiments on the 1H, 13C and 31P n.m.r. of enzyme or 2-phosphoglycollate were combined with the present results to provide the basis for a model in which binding depends on glutamic acid-165 being protonated and on the ligant being fully ionized; additionally, binding affects the ionization of one histidine residue (probably histidine-100). The binding of the glycerol 3-phosphate, on the other hand, was independent of pH over the range pH 6.5--8.5 but decreased at lower pH values. This is explained on a model in which the binding of the monoanion of the ligand is markedly affected by the protonation of a residue in the enzyme, but the binding of the dianion is only slightly affected by this ionization.     

Phosphonomethyl analogues of phosphate ester glycolytic intermediates

Analogues of dihydroxyacetone phosphate and of 3-phosphoglycerate were made in which the phosphate group, –O–PO₃H₂, is replaced by the phosphonomethyl group, –CH₂–PO₃H₂. The analogue of dihydroxyacetone phosphate is a substrate for aldolase and glycerol 1-phosphate dehydrogenase (Stribling, 1974), but not for triose phosphate isomerase. The analogue of 3-phosphoglycerate oxidizes NADH under the combined action of 3-phosphoglycerate kinase and glyceraldehyde 3-phosphate dehydrogenase if ATP is added. Thus four out of the five glycolytic enzymes tested handle the phosphonomethyl compounds like the natural phosphates.     

Metabolism of glycerol by mature boar spermatozoa.

Mature boar spermatozoa oxidized glycerol to carbon dioxide in the absence of any detectable activity of glycerol kinase. With triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase inhibited by the presence of 3-chloro-1-hydroxypropanone (CHOP), dihydroxyacetone phosphate accumulated in incubates when glycerol-3-phosphate was the substrate, but not when it was glycerol. Both dihydroxyacetone and glyceraldehyde could be used as substrates; in the presence of CHOP, dihydroxyacetone phosphate and fructose-1,6-bisphosphate accumulated when dihydroxyacetone was the substrate, but not when it was glyceraldehyde. The metabolic pathways glycerol----glyceraldehyde----glyceraldehyde 3-phosphate and dihydroxyacetone----dihydroxyacetone phosphate have been shown to operate in these cells.     

The histochemical demonstration of fructose diphosphate aldolase activity using a semipermeable membrane technique

Summary In this communication an enzyme histochemical multistep technique for the demonstration of class 1 fructose-1,6-diphosphate aldolase in heart and skeletal muscle sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of the enzyme into the medium during incubation. In the histochemical system the enzyme cleaves the substrate D-fructose-1,6-diphosphate to dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate. The dihydroxyacetone phosphate is reversibly converted into D-glyceraldehyde-3-phosphate by exogenous and endogenous triose phosphate isomerase. Next the D-glyceraldehyde-3-phosphate is oxidized by exogenous and endogenous glyceraldehyde-3-phosphate dehydrogenase and the electrons are transported concomitantly via NAD⁺, phenazine methosulphate and menadione to nitro-BT. Sodium azide and amytal are incorporated to block electron transfer to the cytochromes.     

Enzymes involved in the formation of glycerol 3-phosphate and the by-products dihydroxyacetone and glycerol in Zymomonas mobilis

Abstract In Zymomonas mobilis a novel pathway for the formation of glycerol 3-phosphate was identified by enzymatic studies and nuclear magnetic resonance spectroscopy. This pathway branches off from the Entner-Doudoroff pathway at the intermediate glyceraldehyde 3-phosphate and proceedes via dihydroxyacetone phosphate, dihydroxyacetone, glycerol to glycerol 3-phosphate. The reaction sequence is catalyzed by the enzymes triosephosphate isomerase (0.4 U (mg protein)⁻¹), dihydroxyacetone phosphatase (0.31 U (mg protein)⁻¹), dihydroxyacetone reductase (0.25 U (mg protein)⁻¹), and glycerokinase (0.08 mU (mg protein)⁻¹), respectively. The action of a postulated aldolase catalyzing the cleavage of fructose 6-phosphate to dihydroxyacetone and glyceraldehyde 3-phosphate could be excluded.     

Partial purification, substrate specificity and regulation of alpha-L-glycerolphosphate dehydrogenase from Saccharomyces carlsbergensis.

alpha-L-Glycerolphosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) from Saccharomyces carlsbergensis was purified 400-fold. The enzyme preparation is free of interfering activities, such as glyceraldehyde phosphate dehydrogenase, alcohol dehydrogenase, triose phosphate isomerase and glycerolphosphatase. At pH 7.0 it is specific for NADH (Km = 0.027 mM with 0.8 mM dihydroxyacetone phosphate) and dihydroxyacetone phosphate (Km = 0.2 mM with 0.2 mM NADH). Between pH 5.0 and 6.0 the enzyme functions with NADPH, but only at 7% of the rate with NADH. Various anions (I- greater than SO42- greater than Br- greater than Cl-) act as inhibitors competing with the substrate dihydroxyacetone phosphate. Inorganic phosphate (Ki = 0.1 mM), pyrophosphate and arsenate are strong inhibitors. The nucleotides ATP and ADP are also inhibitory, but their action seems to be of the same type as the general anion competition (Ki = 0.73 mM for ATP). The results are consistent with the notion that the enzyme may regulate the redox potential of the NAD+/NADH couple during fermentation.     

Isolation of a sn-glycerol 3-phosphate: NAD oxidoreductase from spinach leaves.

An NAD-dependent glycerol 3-phosphate dehydrogenase (sn-glycerol 3-phosphate: NAD oxidoreductase; EC 1.1.1.8) has been purified from spinach leaves by a three-step procedure involving ion-exchange, gel filtration, and affinity chromatography. The enzyme has been purified over 10,000-fold to a specific activity of 38. It has a molecular weight of approximately 63,500. The pH optimum for the reduction of dihydroxyacetone phosphate is 6.8 and for glycerol 3-phosphate oxidation it is 9.5. During dihydroxyacetone phosphate reduction hyperbolic kinetics were observed when either NADH or dihydroxyacetone phosphate was the variable substrate, but concentrations of NADH greater than 150 μm were inhibitory. Michaelis constants were 0.30–0.35 mm for dihydroxyacetone phosphate and 0.01 mm for NADH. Glycerol 3-phosphate oxidation obeyed Michaelis-Menten kinetics with a K_m of 0.19 mm for NAD and 1.6 mm for glycerol 3-phosphate. The enzyme was specific for those substrates, and dihydroxyacetone, glyceraldehyde, glyceraldehyde 3-phosphate, NADPH, NADP, and glycerol were not utilized. The spinach leaf enzyme appears to be in the cytoplasm and probably functions for the production of glycerol 3-phosphate from dihydroxyacetone phosphate.     

Stereochemical specificity of the biosynthesis of the alkyl ether bond in alkyl ether lipids.

The stereochemical course of the formation of the alkyl ether bond in alkyl ether lipids was investigated through the synthesis of stereospecifically labeled acyl R- or S-[1-3H]dihydroxyacetone 3-phosphate (DHAP) starting from L-glyceraldehyde. It was demonstrated directly that the formation of the alkyl ether bond results in the stereospecific exchange of the pro-R C-1 hydrogen of DHAP with a proton of water. The configuration of the hydrogen that is retained on C-1 after formation of the alkyl ether bond was also investigated. The alkyl ether lipid was degraded, and the DHAP backbone isolated as glycerol, converted to DHAP via glycerol 3-phosphate and treated with either aldolase or triose phosphate isomerase. The results demonstrated that the retained hydrogen on C-1, which was pro-S in the starting substrate, was pro-S in the product alkyl ether.     

Reaction of triosephosphate isomerase with L-glyceraldehyde 3-phosphate and triose 1,2-enediol 3-phosphate

Triosephosphate isomerase catalyzes the isomerization and/or racemization reactions of L-glyceraldehyde 3-phosphate (LGAP), the enantiomer of the physiological substrate. The reaction is inhibited by the active site directed reagent glycidol phosphate. The amount of protonation product formation catalyzed by a fixed enzyme concentration is nearly independent of increasing steady-state concentrations of triose 1,2-enediol 3-phosphate caused by buffer catalysis of LGAP deprotonation. Therefore, enzymatic protonation of the enediol or enediolate, which could account for the observed enzymatic catalysis of LGAP isomerization and/or racemization, is at best a minor reaction. Instead LGAP reacts directly at the enzyme active site. Triosephosphate isomerase catalysis of the protonation of triose 1,2-enediol 3-phosphate was expected because of the strong evidence supporting an enediol reaction intermediate for the overall reaction catalyzed by isomerase. The most reasonable explanation for the failure to observe enzymatic protonation is that in solution the enediol undergoes beta elimination of phosphate (t 1/2 is estimated to be 10(-6) s) faster than it can diffuse to and form a complex with isomerase.     

Inhibition of triosephosphate isomerase in boar spermatozoa by (S)-3-chlorolactaldehyde

Mature epididymal boar spermatozoa converted fructose and glycerol to carbon dioxide but in the presence of 3-chloro-1-hydroxyacetone these oxidations were inhibited. When the substrate was fructose, there was an increase in the amounts of fructose 1,6-bisphosphate and dihydroxyacetone phosphate but these glycolytic intermediates did not accumulate when glycerol was the substrate. Examination of enzyme activities in mature boar spermatozoa incubated with 3-chloro-1-hydroxyacetone, which is metabolised in vitro to (S)-3-chlorolactaldehyde, confirmed that glyceraldehyde 3-phosphate dehydrogenase and triosephosphate isomerase were both inhibited to equivalent degrees by this metabolite.     

The existence of an electrophilic component in the reaction catalysed by triose phosphate isomerase (Short Communication)

In the presence of triose phosphate isomerase, the substrate dihydroxyacetone phosphate is reduced stereoselectively by NaBH(4). The reduction of enzyme-bound substrate is almost completely or completely stereoselective and occurs about one order of magnitude faster than that in free solution. This acceleration implies a polarization of the carbonyl group when dihydroxyacetone phosphate is bound.     

渗透压在葡萄酒酵母代谢中的调控作用

以磷酸丙糖异构酶部分缺失突变株做对比,研究了渗透压对葡萄酒酵母发酵过程中甘油合成与挥发酸生成的调节作用.结果表明:渗透压对野生型葡萄酒酵母中存在的磷酸二羟丙酮(DHAP)与3-磷酸甘油醛(GA3P)平衡具有调节作用,能使平衡向磷酸二羟丙酮方向迁移以合成更多的甘油,而当磷酸丙糖异构酶部分缺失时渗透压对这一平衡基本不起作用...     

Disequilibrium in the triose phosphate isomerase system in rat liver

1. The equilibrium constant at 38 degrees and I 0.25 of the triose phosphate isomerase reaction was found to be 22.0 and that of the aldolase reaction, 0.99x10(-4)m. The [dihydroxyacetone phosphate]/[glyceraldehyde phosphate] ratio was found to be 9.3 in rat liver. The causes of the apparent deviation of the triose phosphate isomerase system from equilibrium in vivo have been investigated. 2. The equilibria of the triose phosphate isomerase and aldolase reactions were studied with relatively large concentrations of crystalline enzymes and small concentrations of substrates, approximating to those found in rat liver and muscle. There was significant binding of fructose diphosphate by aldolase under these conditions. There was no evidence that binding of glyceraldehyde phosphate by either enzyme affected the equilibria. 3. The deviation from equilibrium of the triose phosphate isomerase system in rat liver can be accounted for by the low activity of the enzyme, in relation to the flux, at low physiological concentrations of glyceraldehyde phosphate (about 3mum). It has been calculated that a flux of 1.8mumoles/min./g. wet weight of liver would be expected to cause the measured degree of disequilibrium found in vivo. 4. The conclusion that the triose phosphate isomerase is not at equilibrium is in accordance with the situation postulated by Rose, Kellermeyer, Stjernholm & Wood (1962) on the basis of isotope-distribution data. 5. The triose phosphate isomerase system is closer to equilibrium in resting muscle probably because of a very low flux and a high enzyme concentration. 6. The aldolase system deviated from equilibrium in rat liver by a factor of about 10 and by a much greater factor in resting muscle. 7. The measurement of total dihydroxyacetone phosphate and glyceraldehyde phosphate content indicates the concentrations of the free metabolites in the tissue. This may not hold for fructose diphosphate, a significant proportion of which may be bound to aldolase.     

NADPH-D-glyceraldehyde 3-phosphate oxidoreductase activity in muscle and other tissues of the rat

Rat muscle was found to contain a NADPH-D-glyceraldehyde 3-phosphate oxidoreductase. Of the ten tissues studied, muscle contained the highest activity at 0.90 ± 0.06 units/g. The activity was not due to L-glycerol 3-phosphate-NAD⁺ oxidoreductase utilising NADPH as a hydrogen donor, nor to coupling of the latter with a NADPH-NAD⁺ oxidoreductase. After partial inhibition of contaminating triose phosphate isomerase with glycidol phosphate, the oxidoreductase rate was faster with glyceraldehyde phosphate than with dihydroxyacetone phosphate as a substrate. Apparent K_m values of 14 μM for D-glyceraldehyde 3-phosphate and 0.7 μM for NADPH were determined.     

Specificity and kinetics of triose phosphate isomerase from chicken muscle

The isolation of crystalline triose phosphate isomerase from chicken breast muscle is described. The values of k(cat.) and K(m) for the reaction in each direction were determined from experiments over wide substrate-concentration ranges, and the reactions were shown to obey simple Michaelis-Menten kinetics. With d-glyceraldehyde 3-phosphate as substrate, k(cat.) is 2.56x10(5)min(-1) and K(m) is 0.47mm; with dihydroxyacetone phosphate as substrate, k(cat.) is 2.59x10(4)min(-1) and K(m) is 0.97mm. The enzyme-catalysed exchange of the methyl hydrogen atoms of the ;virtual substrate' monohydroxyacetone phosphate with solvent (2)H(2)O or (3)H(2)O was shown. This exchange is about 10(4)-fold slower than the corresponding exchange of the C-3 hydrogen of dihydroxyacetone phosphate. The other deoxy substrate, 3-hydroxypropionaldehyde phosphate, was synthesized, but is too unstable in aqueous solution for analogous proton-exchange reactions to be studied.     

Triosephosphate isomerase: removal of a putatively electrophilic histidine residue results in a subtle change in catalytic mechanism

An important active-site residue in the glycolytic enzyme triosephosphate isomerase is His-95, which appears to act as an electrophilic component in catalyzing the enolization of the substrates. With the techniques of site-directed mutagenesis, His-95 has been replaced by Gln in the isomerase from Saccharomyces cerevisiae. The mutant isomerase has been expressed in Escherichia coli strain DF502 and purified to homogeneity. The specific catalytic activity of the mutant enzyme is less than that of wild type by a factor of nearly 400. The mutant enzyme can be resolved from the wild-type isomerase on nondenaturing isoelectric focusing gels, and an isomerase activity stain shows that the observed catalytic activity indeed derives from the mutant protein. The inhibition constants for arsenate and for glycerol phosphate with the mutant enzyme are similar to those with the wild-type isomerase, but the substrate analogues 2-phosphoglycolate and phosphoglycolohydroxamate bind 8- and 35-fold, respectively, more weakly to the mutant isomerase. The mutant enzyme shows the same stereospecificity of proton transfer as the wild type. Tritium exchange experiments similar to those used to define the free energy profile for the wild-type yeast isomerase, together with a new method of analysis involving 14C and 3H doubly labeled substrates, have been used to investigate the energetics of the mutant enzyme catalyzed reaction. When the enzymatic reaction is conducted in tritiated solvent, the mutant isomerase does not catalyze any appreciable exchange between protons of the remaining substrate and those of the solvent either in the forward reaction direction (using dihydroxyacetone phosphate as substrate) or in the reverse direction (using glyceraldehyde phosphate as substrate). However, the specific radioactivity of the product glyceraldehyde phosphate formed in the forward reaction is 31% that of the solvent, while that of the product dihydroxyacetone phosphate formed in the reverse reaction is 24% that of the solvent. The deuterium kinetic isotope effects observed with the mutant isomerase using [1(R)-2H]dihydroxyacetone phosphate and [2-2H]glyceraldehyde 3-phosphate are 2.15 +/- 0.04 and 2.4 +/- 0.1, respectively. These results lead to the conclusion that substitution of Gln for His-95 so impairs the ability of the enzyme to stabilize the reaction intermediate that there is a change in the pathways of proton transfer mediated by the mutant enzyme. The data allow us more closely to define the role of His-95 in the reaction catalyzed by the wild-type enzyme, while forcing us to be alert to subtle changes in mechanistic pathways when mutant enzymes are generated.     

Stabilization of a reaction intermediate as a catalytic device: definition of the functional role of the flexible loop in triosephosphate isomerase

The function of the mobile loop of triosephosphate isomerase has been investigated by deleting four contiguous residues from the part of this loop that interacts directly with the bound substrate. From the crystal structure of the wild-type enzyme, it appears that this excision will not significantly alter the conformation of the rest of the main chain of the protein. The specific catalytic activity of the purified mutant enzyme is nearly 10(5)-fold lower than that of the wild type. Kinetic measurements and isotopic partitioning studies show that the decrease in activity is due to much higher activation barriers for the enolization of enzyme-bound substrate. Although the substrates bind somewhat more weakly to the mutant enzyme than to the wild type, the intermediate analogue phosphoglycolohydroxamate binds much less well (by 200-fold) to the mutant. It seems that the deleted residues of the loop contribute critically to the stabilization of the enediol phosphate intermediate. Consistent with this view, the mutant enzyme can no longer prevent the loss of the enediol phosphate from the active site and its rapid decomposition to methylglyoxal and inorganic phosphate. Indeed, when glyceraldehyde 3-phosphate is the substrate, the enediol phosphate intermediate is lost (and decomposes) 5.5 times faster than it reprotonates to form the product dihydroxyacetone phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mode of action of alpha-chlorohydrin as a male anti-fertility agent. Inhibition of the metabolism of ram spermatozoa by alpha-chlorohydrin and location of block in glycolysis

1. The effect of alpha-chlorohydrin on the metabolism of glycolytic and tricarboxylate-cycle substrates by ram spermatozoa was investigated. The utilization and oxidation of fructose and triose phosphate were much more sensitive to inhibition by alpha-chlorohydrin (0.1-1.0mm) than lactate or pyruvate. Inhibition of glycolysis by alpha-chlorohydrin is concluded to be between triose phosphate and pyruvate formation. Oxidation of glycerol was not as severely inhibited as that of the triose phosphate. This unexpected finding can be explained in terms of competition between glycerol and alpha-chlorohydrin. A second, much less sensitive site, of alpha-chlorohydrin inhibition appears to be associated with production of acetyl-CoA from exogenous and endogenous fatty acids. 2. Measurement of the glycolytic intermediates after incubation of spermatozoal suspensions with 15mm-fructose in the presence of 3mm-alpha-chlorohydrin showed a ;block' in the conversion of glyceraldehyde 3-phosphate into 3-phosphoglycerate. alpha-Chlorohydrin also caused conversion of most of the ATP in spermatozoa into AMP. After incubation with 3mm-alpha-chlorohydrin, glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase activities were decreased by approx. 90% and 80% respectively, and in some experiments aldolase was also inhibited. Other glycolytic enzymes were not affected by a low concentration (0.3mm) of alpha-chlorohydrin. Loss of motility of spermatozoa paralleled the decrease in glyceraldehyde 3-phosphate dehydrogenase activity. alpha-Chlorohydrin, however, did not inhibit glyceraldehyde 3-phosphate dehydrogenase or triose phosphate isomerase in sonicated enzyme preparations when added to the assay cuvette. 3. Measurement of intermediates and glycolytic enzymes in ejaculated spermatozoa before, during and after injection of rams with alpha-chlorohydrin (25mg/kg body wt.) confirmed a severe block in glycolysis in vivo at the site of triose phosphate conversion into 3-phosphoglycerate within 24h of the first injection. Glyceraldehyde 3-phosphate dehydrogenase activity was no longer detectable and both aldolase and triose phosphate isomerase were severely inhibited. Spermatozoal ATP decreased by 92% at this time, being quantitatively converted into AMP. At 1 month after injection of alpha-chlorohydrin glycolytic intermediate concentrations returned to normal in the spermatozoa but ATP was still only 38% of the pre-injection concentration. Motility of spermatozoa was, however, as good as during the pre-injection period. The activity of the inhibited enzymes also returned to normal during the recovery period and 26 days after injection were close to pre-injection values. 4. An unknown metabolic product of alpha-chlorohydrin is suggested to inhibit glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase of spermatozoa. This results in a lower ATP content, motility and fertility of the spermatozoa. Glycidol was shown not to be an active intermediate of alpha-chlorohydrin in vitro.     

Metabolic effects of D-glyceraldehyde in isolated hepatocytes.

The effects of D-glyceraldehyde on the hepatocyte contents of various metabolites were examined and compared with the effects of fructose, glycerol and dihydroxyacetone, which all enter the glycolytic/gluconeogenic pathways at the triose phosphate level. D-Glyceraldehyde (10 MM) caused a substantial depletion of hepatocyte ATP, as did equimolar concentrations of fructose and glycerol. D-Glyceraldehyde and fructose each caused a 2-fold increase in fructose 1,6-bisphosphate and the accumulation of millimolar quantities of fructose 1-phosphate in the cells. D-Glyceraldehyde caused an increase in the glycerol 3-phosphate content and a decrease in the dihydroxyacetone phosphate content, whereas dihydroxyacetone increased the content of both metabolites. The increase in the [glycerol 3-phosphate]/[dihydroxyacetone phosphate] ratio caused by D-glyceraldehyde was not accompanied by a change in the cytoplasmic [NAD+]/[NADH] ratio, as indicated by the unchanged [lactate]/[pyruvate] ratio. The accumulation of fructose 1-phosphate from D-glyceraldehyde and dihydroxyacetone phosphate in the hepatocyte can account for the depletion of the intracellular content of the latter. Presumably ATP is depleted as the result of the accumulation of millimolar amounts of a phosphorylated intermediate, as is the case with fructose and glycerol. It is suggested that the accumulation of fructose 1-phosphate during hepatic fructose metabolism is the result of a temporary increase in the D-glyceraldehyde concentration because of the high rate of fructose phosphorylation compared with triokinase activity. The equilibrium constant of aldolase favours the formation and thus the accumulation of fructose 1-phosphate.     

Metabolic engineering of glycerol production in Saccharomyces cerevisiae

Inactivation of TPI1, the Saccharomyces cerevisiae structural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. In tpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect of tpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, a tpi1delta nde1delta nde2delta gut2delta quadruple mutant was constructed. NDE1 and NDE2 encode isoenzymes of mitochondrial external NADH dehydrogenase; GUT2 encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH in S. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h(-1) at 100 g of glucose. liter(-1)). In aerated batch cultures grown on 400 g of glucose. liter(-1), this engineered S. cerevisiae strain produced over 200 g of glycerol. liter(-1), corresponding to a molar yield of glycerol on glucose close to unity.