Intracellular location of ATP citrate lyase in leaves of Pisum sativum L.

The aim of this work was to discover if there is enough ATP citrate lyase (EC 4.1.3.8) in the cytosol of the leaves of Pisum sativum L. to catalyse the synthesis of the acetyl CoA needed for terpenoid synthesis. Estimates of the maximum catalytic activity of the enzyme in leaves of 7-d-old peas gave values of 113 nmol min^-1 g^-1 fresh weight. The rate of carotenoid accumulation in these leaves corresponded to a requirement for acetyl CoA of 0.7 nmol min^-1 g^-1 fresh weight. The distribution of marker enzymes during fractionation of homogenates of leaves from 7 to 10-d-old peas showed that differential centrifugation led to the isolation in reasonable yields of chloroplasts, mitochondria, peroxisomes and the endomembrane system. None of the above components of the leaf contained appreciable detectable activity of ATP citrate lyase, the distribution of which closely paralleled that of the cytosolic marker. It was concluded that in young leaves of pea most of the ATP citrate lyase is in the cytosol.     

A proposed biochemical mechanism for citric acid accumulation by Aspergillus niger Yang No. 2 growing in solid state fermentation

Aspergillus niger Yang No. 2 and its mutant strain SL1 were grown in solid state fermentation. Samples were taken after 2, 4 and 6 days of incubation and the mycelia were analysed for their intracellular concentrations of some organic acids and adenylates and the activities of selected enzymes. Strain Yang No. 2 contained high concentrations of citrate with very little oxalate, while strain SL1 contained lower concentrations of citrate but considerably higher concentrations of oxalate. As the fermentation proceeded, strain Yang No. 2 showed a much higher ratio of ATP:AMP than did strain SL1. In addition, the enzyme ATP:citrate lyase became undetectable during citrate accumulation in strain Yang No. 2, while its activity remained high during oxalate accumulation in strain SL1. It is proposed that citrate accumulation by strain Yang No. 2 during solid state fermentation is due to blockage of its metabolism in the mitochondrion via inhibition of isocitrate dehydrogenase by the high ATP:AMP ratio, and in the cytosol by repression of ATP:citrate lyase activity.     

Initial steps of sophoroselipid biosynthesis by Candida bombicola ATCC 22214 grown on glucose

 Candida bombicola ATCC 22214 produces the glycolipid sophoroselipid when cultivated on a medium with glucose as the sole carbon source. Under phosphate-limiting conditions the product yield rises from 0.033 to 0.143 and the specific product formation rate rises from 0.004 h^-1 to 0.007 h^-1. Enhanced sophoroselipid synthesis is initiated by the decline of the specific activities of NAD- and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.41 and 1.1.1.42) to 2% and 0% of the initial activities respectively. Constantly high specific activity of citrate synthase (EC 4.1.3.7) causes an accumulation of isocitrate and citrate in the mitochondria. Both acids are transported into the cytosol where citrate is cleaved by ATP: citrate lyase (EC 4.1.3.8) giving rise to acetyl-CoA, the precursor of fatty acid synthesis. The ATP: citrate lyase is unaffected by different energy charges; the apparent K _m values for coenzyme A, ATP and citrate are 23 μM, 250 μM and 256 μM respectively. NADPH for fatty acid synthesis might be generated by further metabolism of oxaloacetate, the other product of the citrate-cleaving reaction, by oxidation of the isocitrate by the cytosolic NADP-dependent isocitrate dehydrogenase or via the hexose monophosphate shunt. A possible explanation for sophoroselipid formation during exponential growth is given. Received: 7 November 1995/Received revision: 19 March 1996/Accepted: 25 March 1996     

Studies on a rat-liver cell-sap protein yielding 3-[32P]-phosphohistidine after incubation with [32P]ATP and alkaline hydrolysis. Identification of the protein as ATP citrate lyase

1. The rat-liver cell-sap material from which 3-[32P]phosphohistidine was previously isolated after incubation with [gamma-32P]ATP and alkaline hydrolysis, was shown to increase about 6-fold on a high-carbohydrate diet. This increase in 32P labelling corresponded to the increase in ATP citrate lyase activity of livers of rats fed on a high-carbohydrate diet, as reported by others. 2. ATP citrate lyase [ATP:citrate oxaloacetate-lyase (CoA-acetylating and ATP-dephopshorylating), EC 4.1.3.8] was purified from rat liver essentially according to the method of Plowman and Cleland (J. Biol. Chem., 242 (1967) 4239). The purified enzyme was incubated for a short time at 0 degree with [gamma-32P]ATP in the presence of 20 mM magnesium acetate. The phosphorylated protein was hydrolysed in alkali and the main part of the radioactivity was identified as 3-[32P]phosphohistidine. The identity of the phosphorylated amino acid was established by Dowex-1 chromatography, paper electrophoresis, paper chromatography and by analysis of the stability to acid. 3. It is concluded from these and previous results from this laboratory that ATP citrate lyase and nucleoside diphosphate kinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) account for most of the normal rat-liver cell-sap protein which is rapidly phosphorylated by ATP.     

Purification and characterization of ATP:citrate lyase from Hydrogenobacter thermophilus TK-6.

ATP:citrate lyase [ATP citrate (pro-3S)-lyase; EC 4.1.3.8] was purified and characterized from the cells of Hydrogenobacter thermophilus, an aerobic, thermophilic, hydrogen-oxidizing bacterium which fixes carbon dioxide by a reductive carboxylic acid cycle. The enzyme was quite stable, even in the absence of sulfhydryl reagents. Optimum pH for reaction was 6.7 to 6.9, and optimum temperature was around 80 degrees C. The molecular weight of native enzyme was estimated to be 260,000 by gel filtration analysis, and that of a subunit was estimated to be 43,000 by sodium dodecyl sulfate-polyacrylamide gel analysis. Km values for reaction components were as follows: citrate, 6.25 mM; ATP, 650 microM; coenzyme A, 40.8 microM; and Mg2+, 8 mM. The enzyme showed citrate synthase activity in the presence of Mg2+, but the reaction rate was very low (less than 1/200 of the lyase activity).     

Calcitonin action on ATP citrate lyase activity in the hepatic cytosol of fed rats and its calcium-calmodulin dependency

The effect of calcitonin (CT) on ATP citrate lyase activity in the hepatic cytosol was investigated after a single subcutaneous administration of the hormone to fed rats. Administration of CT (synthetic [Asu107] eel CT; 80 MRC mU/100 g body weight) produced significant increases in ATP citrate lyase activity and calcium content in the hepatic cytosol of intact and thyroparathyroidectomized rats. Those alterations were also observed with the dose of CT at physiological level. The increased cytosolic ATP citrate lyase activity resulting from CT administration was prevented by treatment with 10 microM EGTA. This enzyme activity was restored by addition of calcium ion (2.5-10 microM). The rise in enzyme activity of CT-treated rats was markedly reduced by the presence of W-7 (10 and 100 microM), a calmodulin inhibitor, in the enzyme assay system, while that of control rats was not significantly altered by the drug. These results suggest that CT increases ATP citrate lyase activity in the hepatic cytosol of fed rats, and that this hormonal regulation may depend on calmodulin, and be mediated through raised calcium in the cytosol.     

Calcitonin and hepatic fatty acid synthesis: effect of thyroparathyroidectomy on the elevation of ATP citrate lyase activity by refeeding of starved rats

The effect of thyroparathyroidectomy (TPTX) on ATP citrate lyase regulation, a rate-limiting enzyme of fatty acid synthesis in hepatic cytosol, was investigated in rats refed after a 24 h fast. ATP citrate lyase activity in the hepatic cytosol was increased 2-fold by refeeding. This increase was suppressed about 50% by TPTX. The suppression of the enzyme activity by TPTX was completely restored by administration of calcitonin (CT; 80 MRC mU/100 g body weight). This hormonal effect was also observed at 20 MRC mU/100 g dose of CT. CT administration to refeeding-TPTX rats produced a significant increase in the calcium content of the liver tissue and the cytosol. The cytosolic ATP citrate lyase activity increase with CT administration was completely blocked by treatment of cytosol with EGTA (10 microM). This inhibition was clearly reversed by addition of calcium ion (1.25-5.0 microM). In addition, CT-induced rise in enzyme activity was markedly reduced by the presence of W-7 (5 and 50 microM), a calmodulin inhibitor, in the enzyme assay system. The present results suggest that CT plays a role in the elevation of hepatic ATP citrate lyase activity brought about by refeeding of fasted rats, and that this hormonal regulation might depend on Ca2+-calmodulin.     

Enhanced docosahexaenoic acid production by reinforcing acetyl-CoA and NADPH supply in <Emphasis Type="Italic">Schizochytrium</Emphasis> sp. HX-308

Docosahexaenoic acid (DHA) production in Schizochytrium sp. HX-308 was evaluated by detecting enzymatic activities of ATP:citrate lyase (EC 4.1.3.8), malic enzyme (EC 1.1.1.40) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) at different fermentation stages. According to the analysis, a regulation strategy was proposed which reinforced acetyl-CoA and NADPH supply at a specific fermentation stage. DHA content of total fatty acids was increased from 35 to 60% by the addition of 4 g/L malic acid at the rapid lipid accumulation stage. Total lipid content also showed an apparent increase of 35% and reached 19 g/L when 40 mL ethanol/L was added at the late lipid accumulation stage.     

ACETYL-CoA SYNTHESIZING ENZYME ACTIVITIES IN SINGLE NERVE CELL BODIES OF RABBIT

Activities of five enzymes (pyruvate dehydrogenase complex; citrate synthase, EC 4.1.3.7; carnitine acetyltransferase, EC 2.3.1.7; acetyl-CoA synthetase, EC 6.2.1.1; and ATP citrate lyase, EC 4.1.3.8) were determined in cell bodies of anterior horn cells and dorsal root ganglion cells from the rabbit. For comparison, molecular layer, granular layer and white matter from rabbit and mouse cerebella and cerebral cortex and striatum from the mouse were analyzed. Samples (3–85 ng dry weight) were assayed in 180 to 370 ml of assay reagents containing CoASH and other substrates in excess. By using ‘CoA cycling’, the assay systems were devised to amplify and measure small amounts of acetyl-CoA formed during the enzyme reactions. Carnitine acetyltransferase was the most active enzyme in single nerve cell bodies and all layer samples, except for rabbit and mouse cerebellar white matter. Citrate synthetase was the lowest in single cell bodies. The activities of carnitine acetyltransferase and acetyl-CoA synthetase (656 and 89.8 mmoles of acetyl-CoA formed/kg of dry weight/h at 38°C) from dorsal root ganglion cells were about 2-fold higher than those from anterior horn cells. The activity of ATP citrate lyase (134mmol of acetyl-CoA formed/kg of dry weight/h at 38°C) from anterior horn cells was approximately twice that from dorsal root ganglion cells. The activity of this enzyme was distributed in a wider range in anterior horn cells than dorsal root ganglion cells. The second highest activity (80.0 mmol of acetyl-CoA formed/kg of dry weight/h at 38°C) of ATP citrate lyase was found in striatum where cholinergic interneurones are abundant. Relatively higher activities of this enzyme were found in cerebellar granular layer and white matter which are known to contain the cholinergic mossy fibers. These results suggested that cholinergic neurones contain higher activity of ATP citrate lyase which is thought to supply acetyl-CoA to choline acetyltransferase (EC 2.3.1.6) as a substrate to form acetylcholine.     

Compartmentation of enzymes: ATP citrate lyase in hepatocytes from fed or fasted rats

Compared with traditional techniques of tissue homogenization, digitonin fractionation of isolated hepatocytes provides a much more rapid and, in some instances, more accurate determination of enzyme compartmentation. Results with ATP citrate lyase (EC 4.1.3.8) illustrate the information that uniquely can be obtained. Although the enzyme was previously thought to be entirely cytosolic, digitonin fractionation has shown that a portion of total cellular ATP citrate lyase is bound to mitochondria or some other structure, and the amount bound varies with the animal's nutritional state. In hepatocytes from rats that were starved for 2 days, fed NIH stock diet ab libitum, or starved for 2 days and then refed a fat-free diet for 2 days, the noncytosolic activity was, respectively, 52, 21, or 24% of total cellular lyase. However, because starvation/refeeding greatly induces lipogenic enzymes, the amount of bound lyase activity in this dietary state was 10-12 times greater than that in rats that were starved or fed ad libitum. The association of citrate lyase with a subcellular organelle is also influenced by CoA. Addition of 20 microM CoA to the digitonin fractionation medium caused all of the lyase to be released from cells like a cytosolic enzyme. Conversely, when cellular free CoA was decreased by incubating hepatocytes with the hypolipidemic agent 5-(tetradecyloxy)-2-furoic acid, the amount of bound lyase was increased. These results suggest the possibility that the noncytosolic ATP citrate lyase may have a special role in lipogenesis.     

Acetate oxidation to CO2 via a citric acid cycle involving an ATP-citrate lyase: a mechanism for the synthesis of ATP via substrate level phosphorylation in Desulfobacter postgatei growing on acetate and sulfate

Desulfobacter postgatei is an acetate-oxidizing, sulfate-reducing bacterium that metabolizes acetate via the citric acid cycle. The organism has been reported to contain a si-citrate synthase (EC 4.1.3.7) which is activated by AMP and inorganic phosphate. It is show now, that the enzyme mediating citrate formation is an ATP-citrate lyase (EC 4.1.3.8) rather than a citrate synthase. Cell extracts (160,000xg supernatant) catalyzed the conversion of oxaloacetate (apparent K _m=0.2 mM), acetyl-CoA (app. K _m=0.1 mM), ADP (app. K _m=0.06 mM) and phosphate (app. K _m=0.7 mM) to citrate, CoA and ATP with a specific activity of 0.3 mol·min^-1·mg^-1 protein. Per mol citrate formed 1 mol of ATP was generated. Cleavage of citrate (app. K _m=0.05 mM; V _max=1.2 mol · min^-1 · mg^-1 protein) was dependent on ATP (app. K _m=0.4 mM) and CoA (app. K _m=0.05 mM) and yielded oxaloacetate, acetyl-CoA, ADP, and phosphate as products in a stoichiometry of citrate:CoA:oxaloacetate:ADP=1:1:1:1. The use of an ATP-citrate lyase in the citric acid cycle enables D. postgatei to couple the oxidation of acetate to 2 CO₂ with the net synthesis of ATP via substrate level phosphorylation.     

Citrate lyase from Streptococcus diacetilactis

Citrate lyase (EC 4.1.3.6) was purified 38-fold from cell-free extracts of Streptococcus diacetilactis. The enzyme was homogeneous in analytical ultracentrifugation and polyacrylamide gel electrophoresis The final enzyme preparation contained acetate: HS-citrate lyase ligase—an acetylating enzyme which converts inactive HS-citrate lyase into enzymatically active acetyl-S-citrate lyase. This enzyme activity was purified 25-fold over the crude extract and seemed to be associated with citrate lyase. Partially purified citrate lyase from Leuconostoc citrovorum contained also its acetylating enzyme. Purified citrate lyases from Klebsiella aerogenes and Rhodopseudomonas gelatinosa were devoid of acetylating enzyme activity. The HS-form of citrate lyase from S. diacetilactis was completely acetylated and hence activated by incubation with ATP and acetate for 25 min at 25° C. The enzyme did not acetylate the HS-lyases from R. gelatinosa and K. aerogenes. In contrast to the citrate lyases from R. gelatinosa and K. aerogenes the enzymes from S. diacetilactis and L. citrovorum showed onlya very weak reaction inactivation. It is assumed that this is due to the association of the acetylating enzymes with these lyases.     

Phosphorylation of ATP citrate lyase in response to glucagon.

Incubation of hepatocytes with [32P]orthophosphate resulted in the incorporation of 32P into material that is precipitated by reaction with antibodies to ATP citrate lyase. The amount of radioactivity precipitated was decreased when unlabeled, purified ATP citrate lyase was added to extracts of hepatocytes that had been incubated with [32P]orthophosphate. Addition of glucagon to hepatocytes that had been preincubated with [32P]orthophosphate resulted in a 56% increase in acid-stable 32P in the trichloroacetic acid-insoluble portion of immunoprecipitates. Catalytic phosphate bound to ATP citrate lyase reaction with ATP and Mg2+ is acid-labile; thus, glucagon-dependent phosphorylation is distinguished from the catalytic phosphate. When hepatocytes were incubated in the absence of [32P]orthophosphate and extracted in a medium containing [gamma-32P]ATP, no acid-stable 32P was present in immunoprecipitates. This indicates that the incorporation into ATP citrate lyase of acid-stable phosphate occurs prior to extraction of the enzyme. Preliminary studies, using a procedure that allows for measurement of enzyme activity starting 1 min after beginning the extraction of lyase from hepatocytes, have shown no difference in lyase activity when hepatocytes are treated with or without glucagon.     

Apparent Inhibition of ATP Citrate Lyase by l-Glutamate In Vitro Is Due to the Presence of Glutamine Synthetase

Preincubation in assay mixture for 30 min at 37 degrees C of ATP citrate lyase from rat brain and liver results in 65-70% inhibition in the presence of 10 mM L-glutamate. This inhibition is specific since none of the known brain metabolites of glutamate shows this effect. ATP and ammonium sulphate-suspended, commercially purified malate dehydrogenase are both important in the generation of inhibition; citrate and NADH are not. The ATP citrate lyase activity in desalted crude extracts and 11% polyethylene glycol-precipitated fractions is inhibited but the enzyme purified by dye affinity chromatography is unaffected. Such purification reveals the presence of a factor responsible for the generation of the inhibition shown to be of Mr 380,000. These lines of evidence implicate endogenous glutamine synthetase, and the involvement of this enzyme is established by the use of its inhibitor L-methionine sulphoximine and by the addition of purified glutamine synthetase to restore the glutamate inhibition of purified ATP citrate lyase. The phenomenon probably arises from the production by glutamine synthetase of ADP, a known product inhibitor of ATP citrate lyase. Therefore contrary to previous reports elsewhere, L-glutamate has no role in the regulation of brain ATP citrate lyase and thus the supply of cytoplasmic acetyl groups for biosynthesis.     

Distribution of the ability for citrate utilization amongst Clostridia

44 species of the genus Clostridium were examined with regard to their ability to grow on citrate. In addition to Clostridium sphenoides, a known citrate utilizer, the following species were found to utilize citrate: C. sporosphaeroides, C. symbiosum, C. rectum, C. indolis, C. subterminale and C. sporogenes. The major products formed from citrate were acetate, ethanol and carbon dioxide (not measured). Minor products were butyrate, butanol, acetone and acetoin.The enzyme citrate lyase was detectable in cell extracts of C. sporosphaeroides and C. symbiosum using an optical assay. Evidence for the presence of this enzyme in the other species was obtained in immunological experiments and in experiments with [1,5-¹⁴C]citrate.     

Pathways to acetyl-CoA formation in Candida albicans

The supply of acetyl units from the mitochondrion to the cytosol of Candida albicans appears to be dependent only upon the activity of carnitine acetyltransferase (CAT). The enzyme ATP:citrate lyase (ACL), the major source of acetyl units in oleaginous yeasts, is absent from C. albicans in both the mycelial and yeast forms. There appears to be no other active translocation of acetate or acetyl groups except via the action of carnitine acetyltransferase.     

Enzyme activities in oleaginous yeasts accumulating and utilizing exogenous or endogenous lipids

The activities of ATP: citrate lyase (ACL; EC 4. 1. 3. 8), carnitine acetyltransferase (CAT; EC 2. 3. 1. 7), NADP+-dependent isocitrate dehydrogenase (ICDH; EC 1. 1. 1. 42), isocitrate lyase (ICL; EC 4. 1. 3. 1) and malic enzyme (malate dehydrogenase; EC 1. 1. 1. 40) were measured in four oleaginous yeasts, Candida curvata D, Trichosporon cutaneum and two strains of Rhodosporidium toruloides, grown either to accumulate lipid, or to utilize their own lipid reserves or an exogenous supply of lipid. During lipid utilization, activities of ACL and malic enzyme diminished to low levels; CAT and ICL increased considerably in activity and ICDH activity was slightly increased but catalase (EC 1. 11. 1. 6) diminished in activity in both strains of R. toruloides. In all cases, yeasts utilizing exogenous lipid showed greater changes in enzyme activities than cells utilizing their endogenous reserves. Electron micrographs of Candida curvata D showed proliferation of peroxisomes in starved cells utilizing their own lipid reserve though peroxisomes were more in evidence when the yeast had been grown on exogenous lipid. In Lipomyces starkeyi, which shows only minimal utilization of its stored lipid and furthermore cannot grow on exogenous lipid, only the occasional peroxisome was seen when cells were starved of carbon.     

Factors involved in changes in hepatic lipogenesis during development of the rat

1. Changes in the activities of acetyl-CoA carboxylase (EC 6.4.1.2), phosphofructokinase (EC 2.7.1.11), aldolase (EC 4.1.2.13), extramitochondrial aconitate hydratase (EC 4.2.1.3) and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) have been measured in the livers of developing rats from late foetal life to maturity. 2. The effect of altering the weaning time on some enzymes associated with lipogenesis has been studied. Weaning rats at 15 days of age instead of 21 days results in an immediate increase in the activity of ;malic' enzyme (EC 1.1.1.40) whereas the activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and ATP citrate lyase (EC 4.1.3.8) did not increase until 4-5 days and acetyl-CoA carboxylase 2-3 days after early weaning. Weaning rats on to an artificial-milk diet led to complete repression of the rise in activity of hepatic enzymes associated with lipogenesis normally found on weaning, except for ;malic' enzyme, which increased in activity after 20 days of age. 3. The effect of intraperitoneal injections of glucagon, cortisol, growth hormone and thyroxine on the same hepatic enzymes has been investigated. Only thyroxine had any effect on enzyme activities and caused a 20-fold increase in ;malic' enzyme activity and a twofold increase in ATP citrate lyase activity. 4. The activities of hepatic glucose 6-phosphate dehydrogenase and ;malic' enzyme are higher in adult female than in adult male rats and it has been shown that this sex difference in enzyme activities is due to both male and female sex hormones. 5. Hepatic malate, citrate, pyruvate, glucose 6-phosphate and phosphoenolpyruvate concentrations have been measured throughout development. 6. The results are discussed in relation to the dietary and hormonal control of hepatic enzyme activities during development.     

Characterization of citrate lyase from Clostridium sporosphaeroides

Cells of Clostridium sporosphaeroides which were grown on citrate contained citrate lyase and citrate lyase acetylating enzyme, but no detectable citrate synthase and citrate lyase deacetylase activities. Citrate lyase from C. sporosphaeroides was purified to homogeneity as judged by polyacrylamide gel electrophoresis and high performance liquid chromatography. In contrast to the enzyme from Clostridium sphenoides, the addition of l-glutamate was not necessary for activity and stabilization of the enzyme. The purified enzyme had a specific activity of 34 U/mg protein and was comparable to other citrate lyases with respect to its molecular weight and subunit composition. Electron microscopic investigations showed that similar to the lyase from C. sphenoides and in contrast to all other citrate lyases examined so far, the majority of the enzyme molecules was present in star form.     

Synthesis of citrate from phosphoenolpyruvate and acetylcarnitine by mitochondria from rabbit, pigeon and rat liver: Implications for lipogenesis

Rabbit, pigeon and rat liver mitochondria convert exogenous phosphoenolpyruvate and acetylcarnitine to citrate at rates of 14, 74 and 8 nmol/15 min/mg protein. Citrate formation is dependent on exogenous HCO3, is increased consistently by exogenous nucleotides (GDP, IDP, GTP, ADP, ATP) and inhibited strongly by 3-mercaptopicolinate and 1,2,3-benzenetricar☐ylate. Citrate is not made from pyruvate alone or combined with acetylcarnitine. Pigeon and rat liver mitochondria make large amounts of citrate from exogenous succinate, suggesting the presence of an endogenous source of acetyl units or a means of converting oxalacetate to acetyl units. Citrate synthesis from succinate by pigeon and rabbit mitochondria is increased significantly by exogenous acetylcarnitine. Pigeon and rat liver contain 80 and 15 times, respectively, more ATP:citrate lyase activity than does rabbit liver. Data suggest that mitochondrial phosphoenolpyruvate car☐ykinasein vivo could convert glycolysis-derived phosphoenolpyruvate to oxalacetate that, with acetyl CoA, could form citrate for export to support cytosolic lipogenesis as an activator of acetyl CoA car☐ylase, a carbon source via ATP:citrate lyase and NADPH via NADP: malate dehydrogenase or NADP: isocitrate dehydrogenase.