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PubMed2003

Acetohydroxyacid synthase: a new enzyme for chiral synthesis of R-phenylacetylcarbinol.

We have found that acetohydroxyacid synthase (AHAS) is an efficient catalyst for the enantiospecific (> or =98% enantiomeric excess) synthesis of (R)-phenylacetylcarbinol (R-PAC) from pyruvate and benzaldehyde, despite the fact that its normal physiological role is synthesis of (S)-acetohydroxyacids from pyruvate and a second ketoacid. (R)-phenylacetylcarbinol is the precursor of important drugs having alpha and beta adrenergic properties, such as L-ephedrine, pseudoephedrine, and norephedrin. It is currently produced by whole-cell fermentations, but the use of the isolated enzyme pyruvate decarboxylase (PDC) for this purpose is the subject of active research and development efforts. Some of the AHAS isozymes of Escherichia coli have important advantages compared to PDC, including negligible acetaldehyde formation and high conversion of substrates (both pyruvate and benzaldehyde) to PAC. Acetohydroxyacid synthase isozyme I is particularly efficient. The reaction is not limited to condensation of pyruvate with benzaldehyde and other aromatic aldehydes may be used.

Acetolactate Synthase↗
PubMed1998

Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: IV. Enzymes and fluxes of pyruvate metabolism.

The activities of pyruvate kinase (PK), pyruvate: formate-lyase (PFL), pyruvate dehydrogenase (PDH), and citrate synthase (CS) involved in the anaerobic glycerol conversion by Klebsiella pneumoniae were studied in continuous culture under conditions of steady states and sustained oscillations. Both the in vitro and in vivo activities of PK, PFL, and PDH are strongly affected by the substrate concentration and its uptake rate, as is the in vitro activity of CS. The flux from phosphoenolpyruvate to pyruvate is found to be mainly regulated on a genetic level by the synthesis rate of PK, particularly at low substrate concentration and low growth rate. In contrast, the conversion of pyruvate to acetyl-CoA is mainly regulated on a metabolic level by the in vivo activities of PFL and PDH. The ratio of in vitro to in vivo activities is in the range of 1 to 1.5 for PK, 5 to 17 for PFL and 5 to 80 for PDH under the experimental conditions. The regulation of in vivo activity and synthesis of these enzymes is sensitive to fluctuations of culture conditions, leading to oscillations of both the in vitro and in vivo activities. In particular, PFL is strongly affected during oscillations; its average in vitro activity is only about half of its corresponding steady-state value under similar environmental conditions. The average in vitro activities of PDH and PK under oscillations are close to their corresponding steady-state values. In contrast to all other enzymes measured for the glycerol metabolism by K. pneumoniae PFL and PDH are more effectively in vivo utilized under oscillations than under steady state, underlining the peculiar role of pyruvate metabolism in the dynamic responses of the culture.

Acetyltransferases↗
PubMed1995

Heterogeneous responses to an intravenous glucose load.

The aim of this study was to determine how the insulin sensitive enzymes pyruvate dehydrogenase (PDH) complex and glycogen synthase (GS) of different tissues respond to an endogenous pulse of insulin elicited by an intravenous infusion of glucose. An infusion of glucose (0.5 g/kg) into conscious, unrestrained animals via an indwelling cannula rapidly elevated plasma insulin concentration (to approx. 600 microU/ml after 10 min). The animals were sacrificed at selected time points after the commencement of infusion. Samples of heart, red quadriceps muscle, white adipose tissue (WAT) and brown adipose tissue (BAT) were excised and assayed for PDH complex and GS activities. The glucose infusion elicited a rapid (< 5-10 min) increase in both PDH complex and GS activities in heart, BAT and WAT. The maximum rise in the activity of PDH and GS above basal were (respectively) 2- and 8-fold for heart, 5.5- and 5-fold for BAT, and 3.5- and 4-fold for WAT. The return of PDH complex activity to basal values was also very rapid (occurring over the next 20 min). The glucose infusion also stimulated GS activity in red quadriceps muscle but was, however without effect on PDH complex activity in this tissue. We conclude that although insulin stimulates PDH and GS with the same time course and magnitude in many insulin sensitive tissues, the time course and magnitude of insulin stimulation of these enzymes can vary between tissues. These results may mean that the stimulation of PDH complex and GS by insulin occurs via different receptor-effector pathways.

Adipose Tissue↗
PubMed1986

Skeletal muscle enzyme activities in healthy young subjects.

In three groups of healthy young subjects (n = 33; mean ages 6.4, 13.5, 17.1 years), muscle enzyme activities (creatine kinase, hexose phosphate isomerase, aldolase, pyruvate kinase, lactate dehydrogenase, citrate synthase, fumarase) of the vastus lateralis muscle were investigated to show age-dependent variations. A significant age-dependent increase in aldolase (P less than 0.05) and pyruvate kinase (P less than 0.01) activity and a decrease in fumarase activity (P less than 0.01) were computed. In relation to the age-dependent variation, maximum LDH activities could be measured at an age of 12-14 years; significantly decreased activities of the glycolytic enzymes could only be found in the youngest group.

Adolescent↗
PubMed1995

Modifications by chronic intermittent hypoxia and drug treatment on skeletal muscle metabolism.

The energy metabolism was evaluated in gastrocnemius muscle from 3-month-old rats subjected to either mild or severe 4-week intermittent normobaric hypoxia. Furthermore, 4-week treatment with CNS-acting drugs, namely, alpha-adrenergic (delta-yohimbine), vasodilator (papaverine, pinacidil), or oxygen-increasing (almitrine) agents was performed. The muscular concentration of the following metabolites was evaluated: glycogen, glucose, glucose 6-phosphate, pyruvate, lactate, lactate-to-pyruvate ratio; citrate, alpha-ketoglutarate, succinate, malate; aspartate, glutamate, alanine; ammonia; ATP, ADP, AMP, creatine phosphate. Furthermore the Vmax of the following muscular enzymes was evaluated: hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase; citrate synthase, malate dehydrogenase; total NADH cytochrome c reductase; cytochrome oxidase. The adaptation to chronic intermittent normobaric mild or severe hypoxia induced alterations of the components in the anaerobic glycolytic pathway [as supported by the increased activity of lactate dehydrogenase and/or hexokinase, resulting in the decreased glycolytic substrate concentration consistent with the increased lactate production and lactate-to-pyruvate ratio] and in the mitochondrial mechanism [as supported by the decreased activity of malate dehydrogenase and/or citrate synthase resulting in the decreased concentration of some key components in the tricarboxylic acid cycle]. The effect of the concomitant pharmacological treatment suggests that the action of CNS-acting drugs could be also related to their direct influence on the muscular biochemical mechanisms linked to energy transduction.

Adenine Nucleotides↗
PubMed1995

N-acetyl-D-neuraminic acid synthesis in Escherichia coli K1 occurs through condensation of N-acetyl-D-mannosamine and pyruvate.

Two enzymes have been found to be involved in bacterial N-acetyl-D-neuraminic acid (NeuAc) synthesis: NeuAc synthase, which condenses N-acetyl-L,D-mannosamine and phosphoenolpyruvate, and NeuAc lyase or NeuAc aldolase, which condenses N-acetyl-D-mannosamine and pyruvate. When we used Escherichia coli K1 crude extracts, we observed the generation of NeuAc in the presence of N-acetylmannosamine and both phosphoenolpyruvate (NeuAc synthase activity) or pyruvate (NeuAc lyase activity). However, when crude extracts were fractionated by Sephacryl S-200 chromatography, NeuAc synthase activity disappeared. A chromatographic peak of NeuAc synthase activity was detected when column fractions were re-tested in the presence of the active NeuAc lyase peak. Furthermore, crude extracts converted phosphoenolpyruvate into pyruvate. Pyruvate depletion, due to the addition of pyruvate decarboxylase to the NeuAc synthase reaction mixture, blocked NeuAc formation. Moreover, after NeuAc lyase immunoprecipitation no NeuAc synthase was detected. These findings suggest that NeuAc synthase is not present in E. coli K1 and therefore that NeuAc lyase is the only enzyme responsible for NeuAc synthesis in this bacterium.

Escherichia coli↗
PubMed1987

Effects of chronic administration of dextroamphetamine on enzymes of energy metabolism in regions of the rat brain.

In the present study the effects of chronic administration of dextroamphetamine on energy metabolism in the brain of the rat were examined. The enzymes studied were: hexokinase (soluble and particulate forms), phosphofructokinase, pyruvate kinase, lactate dehydrogenase, citrate synthase, NAD+ and NADP+-dependent isocitrate dehydrogenases, succinate dehydrogenase and malate dehydrogenase. All the activities of the enzymes were assayed in four regions of the brain of the rat (cerebellum, medulla oblongata and pons, cererbral cortex and diencephalon). Rats were injected intaperitoneally once daily with dextroamphetamine for 20 consecutive days. The initial dose was 5 mg/kg/day and the dose was then increased by 1 mg/kg/every 5 days up to a total of 8 mg/kg/day on days 16-20. In the glycolytic enzymes a reduction of the activity of phosphofructokinase was found in the diencephalon and an increase of the activity of pyruvate kinase and lactate dehydrogenase in the diencephalon and medulla oblongata and pons, respectively. Citrate synthase was the only enzyme in the Krebs' cycle affected by chronic administration of dextroamphetamine. The results presented here show that chronic administration of dextroamphetamine produced important changes in some enzymes of glycolysis and the Krebs' cycle in the brain of the rat.

Animals↗
PubMed1996

Interaction of carbohydrate and fat fuels in human skeletal muscle: impact of obesity and NIDDM.

The current study was undertaken to examine the impact that obesity and non-insulin-dependent diabetes mellitus (NIDDM) have on the ability of glucose to stimulate its own uptake and oxidation in muscle. Euglycemic and hyperglycemic clamp experiments were performed with somatostatin infusions so that insulin could be replaced to basal levels or to physiological hyperinsulinemia. Arteriovenous leg balance methods were used to measure the pathways of leg muscle glucose uptake, oxidation, and storage. Percutaneous biopsies of the vastus lateralis muscle were taken to determine the pyruvate dehydrogenase complex or glycogen synthase activities. During basal insulin replacement, obese compared with lean nondiabetic subjects had higher values for glucose uptake, respiratory quotient, and glucose oxidation (all P<0.05) and a higher proportion of leg energy expenditure derived from glucose. Obese NIDD patients had a greater reliance on fat calories than lean diabetics during basal insulin replacement (P< 0.05). Hyperinsulinemia increased leg glucose metabolism (P<0.001) in all groups, but obese NIDD patients were significantly more insulin resistant. Hyperglycemia in NIDDM compensated for insulin resistance to the extent that rates of glucose metabolism were the same as those for nondiabetics studied at euglycemia. When nondiabetics were studied at hyperglycemia matched to the diabetics, the insulin resistance was still readily apparent.

Adult↗
PubMed2005

Role of arginine 138 in the catalysis and regulation of Escherichia coli dihydrodipicolinate synthase.

In plants and bacteria, the branch point of (S)-lysine biosynthesis is the condensation of (S)-aspartate-beta-semialdehyde [(S)-ASA] and pyruvate, a reaction catalyzed by dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52). It has been proposed that Arg138, a residue situated at the entrance to the active site of DHDPS, is responsible for binding the carboxyl of (S)-ASA and may additionally be involved in the mechanism of (S)-lysine inhibition. This study tests these assertions by mutation of Arg138 to both histidine and alanine. Following purification, DHDPS-R138H and DHDPS-R138A each showed severely compromised activity (approximately 0.1% that of the wild type), and the apparent Michaelis-Menten constant for (S)-ASA in each mutant, calculated using a pseudo-single substrate analysis, was significantly higher than that of the wild type. This provides good evidence that Arg138 is indeed essential for catalysis and plays a key role in substrate binding. To test whether structural changes could account for the change in kinetic behavior, the solution structure was probed via far-UV circular dichroism, confirming that the mutations at position 138 did not modify secondary structure. The crystal structures of both mutant enzymes were determined, confirming the presence of the mutations and suggesting that Arg138 plays an important role in catalysis: the stabilization of the catalytic triad residues, a motif we have previously demonstrated to be essential for activity. In addition, the role of Arg138 in (S)-lysine inhibition was examined. Both mutant enzymes showed the same IC(50) values as the wild type but different partial inhibition patterns, from which it is concluded that arginine 138 is not essential for (S)-lysine inhibition.

Arginine↗
PubMed2005

Pyruvate modulates hepatic mitochondrial functions and reduces apoptosis indicators during hemorrhagic shock in rats.

BACKGROUND: Dysfunctional mitochondria have been widely accepted as one of the key targets and a mediator of secondary cell injury and organ failure during hemorrhagic shock (HS). The liver is known to be the first organ to display the signs of injury during HS. This report describes experiments to determine whether modulation of hepatic mitochondrial dysfunctions by pharmacologic agents could prevent liver injury in rats subjected to HS. METHODS: In this study, Sprague-Dawley rats were either treated as controls or subjected to computer-controlled arterial hemorrhage (40 mmHg) for 60 min followed by resuscitation with hypertonic saline, hypertonic beta-hydroxybutyrate, or hypertonic sodium pyruvate for the next 60 min before death. During the course of the experiment, animals were continuously monitored for hemodynamic and metabolic parameters. At the end of the experiment, the liver was excised and examined for oxidative injury, mitochondrial functions, expression of nitric oxide synthase, and indicators of apoptosis. RESULTS: In comparison to hypertonic saline and hypertonic beta-hydroxybutyrate, pyruvate significantly protected the liver from oxidative injury, prevented the up-regulation of nitric oxide synthase, inhibited pyruvate dehydrogenase deactivation, and improved cellular energy charge and mitochondrial functions. In addition, pyruvate also reduced cleavage of poly-adenosine diphosphate ribose polymerase by preventing leakage of mitochondrial cytochrome c in the liver of HS animals. CONCLUSIONS: These data suggest that modulation of mitochondrial metabolic functions is likely to be one of the important mechanisms by which pyruvate exerts its protective effects on the liver during HS and resuscitation in rats.

Animals↗
PubMed1994

Coordinate regulation of glycolytic and lipogenic gene expression by polyunsaturated fatty acids.

Using a combination of in vivo and in vitro studies, we have investigated the impact of polyunsaturated fatty acids (PUFA) on the expression of several genes encoding proteins involved in hepatic glycolysis and lipogenesis. Meal-training rats to a high glucose diet containing 10% triolein led to a significant induction of hepatic mRNAs encoding glucokinase (GK), pyruvate kinase (PK), fatty acid synthase (FAS), malic enzyme (ME), and the S14 protein (S14), but had no effect on thyroid hormone receptor-beta 1 (TR beta 1) and c/EBP alpha gene expression. Replacing triolein with menhaden oil attenuated (by 50-90%) the induction of mRNA encoding GK, ME, PK, FAS, and S14. This effect was rapid (within hours) and for FAS and S14, directed at the transcriptional level. The mRNAs encoding TR beta 1, c/EBP alpha and beta-actin were unaffected by menhaden oil. Studies with cultured primary hepatocytes showed that C18:3,omega 3 (n-3), C18:3,omega 6 (n-6), C20:4, omega 6 (n-6), and C20:5,omega 3 (n-3) were all equally effective at suppressing the level of mRNAs encoding FAS, S14, and PK. This effect was specific for glycolytic and lipogenic enzymes, as expression of beta-actin was not affected by these fatty acids. Moreover, the fatty acids had only marginal effects on cell viability as judged by lactate dehydrogenase release. These data indicate that polyunsaturated fatty acids coordinately regulate the expression of several enzymes involved in carbohydrate and lipid metabolism. The mechanism of control does not require extrahepatic factors or fatty acid metabolism.

Animals↗
PubMed1989

Effect of nutritional status on insulin sensitivity in vivo and tissue enzyme activities in the rat.

The hyperinsulinaemic-glucose-clamp technique, in combination with measurement of glucose turnover in conscious unrestrained rats, was used to assess the effects of nutritional status on insulin sensitivity in vivo and glucose metabolism. Liver, heart and quadriceps skeletal-muscle glycogen content and activities of pyruvate dehydrogenase (PDH) and glycogen synthase were measured both basally and at the end of a 2.5 h glucose clamp (insulin 85 munits/h) in rats 6, 24 and 48 h after food withdrawal. Clamp glucose requirement and glucose turnover were unchanged by fasting. Activation of glycogen synthase and glycogen deposition in liver and skeletal muscle during the clamps were also not impaired in rats after a prolonged fast. By contrast with skeletal muscle, activation of cardiac-muscle glycogen synthase and glycogen deposition during the clamps were markedly impaired by 24 h of fasting and were undetectable at 48 h. Skeletal-muscle PDH activity fell with more prolonged fasting (6 h, 15.3 +/- 3.4%; 24 h, 4.7 +/- 0.7%; 48 h, 4.3 +/- 0.6% active; P less than 0.005), but at 24 and 48 h was stimulated by the clamp to values unchanged by the duration of fasting. Stimulation of cardiac PDH activity by the clamp was, however, impaired in rats fasted for 24 or 48 h. Basal hepatic PDH did not change significantly with fasting (6 h, 5.3 +/- 1.1%; 24 h, 4.6 +/- 0.7%; 48 h, 3.9 +/- 0.5%), and, although it could be partly restored at 24 h, very little stimulation occurred at 48 h. Hepatic pyruvate kinase and acetyl-CoA carboxylase activity were both stimulated by the clamps, and this was not impaired with more prolonged fasting. During the glucose clamps, blood concentrations of lactate, pyruvate and alanine were increased to a greater extent in rats fasted for 24 and 48 h than in rats studied 6 h after food withdrawal. The findings suggest that, although sensitivity to insulin of whole-body glucose disposal is unchanged with fasting, there may be qualitative differences in the metabolism of glucose.

Animals↗
PubMed1996

Insulin/glucose-, pyruvate- and polyunsaturated fatty acid-responsive region(s) of rat fatty acid synthase gene promoter.

To investigate the regulatory DNA sequences required for polyunsaturated fatty acid (PUFA)-suppression of fatty acid synthase (FAS) gene as well as for insulin and/or carbohydrate-stimulation of this gene, primary hepatocytes were transfected with plasmids containing the 5'-flanking sequence of the rat FAS gene fused to the CAT gene. Sequences from -1604, -88 or -57 to +79 of the FAS gene directed an increase in CAT activity in the hepatocytes when insulin/glucose was added to the medium, in accordance with the responses on the endogenous FAS gene expression. The CAT activities were reduced by the addition of PUFA. Further deletion to -34, however, resulted in loss of the responses. The results suggest that the region from -57 to -34 of the FAS gene may be responsible for regulation due to insulin/glucose and PUFAs. Moreover, the region was also responsible for stimulation due to pyruvate alone.

Animals↗
PubMed2000

Characterization of the role of AMP-activated protein kinase in the regulation of glucose-activated gene expression using constitutively active and dominant negative forms of the kinase.

In the liver, glucose induces the expression of a number of genes involved in glucose and lipid metabolism, e.g., those encoding L-type pyruvate kinase and fatty acid synthase. Recent evidence has indicated a role for the AMP-activated protein kinase (AMPK) in the inhibition of glucose-activated gene expression in hepatocytes. It remains unclear, however, whether AMPK is involved in the glucose induction of these genes. In order to study further the role of AMPK in regulating gene expression, we have generated two mutant forms of AMPK. One of these (alpha1(312)) acts as a constitutively active kinase, while the other (alpha1DN) acts as a dominant negative inhibitor of endogenous AMPK. We have used adenovirus-mediated gene transfer to express these mutants in primary rat hepatocytes in culture in order to determine their effect on AMPK activity and the transcription of glucose-activated genes. Expression of alpha1(312) increased AMPK activity in hepatocytes and blocked completely the induction of a number of glucose-activated genes in response to 25 mM glucose. This effect is similar to that observed following activation of AMPK by 5-amino-imidazolecarboxamide riboside. Expression of alpha1DN markedly inhibited both basal and stimulated activity of endogenous AMPK but had no effect on the transcription of glucose-activated genes. Our results suggest that AMPK is involved in the inhibition of glucose-activated gene expression but not in the induction pathway. This study demonstrates that the two mutants we have described will provide valuable tools for studying the wider physiological role of AMPK.

AMP-Activated Protein Kinases↗
PubMed2001

Mitochondria control ampa/kainate receptor-induced cytoplasmic calcium deregulation in rat cerebellar granule cells.

Although mitochondria mediate the delayed failure of cytoplasmic Ca(2+) homeostasis [delayed Ca(2+) deregulation (DCD)] in rat cerebellar granule cells resulting from chronic activation of NMDA receptors, their role in AMPA/KA-induced DCD remains to be established. The mitochondrial ATP synthase inhibitor oligomycin protected cells against KA- but not NMDA-evoked DCD. In contrast to NMDA-evoked DCD, no additional protection was afforded by the further addition of rotenone. The effects of KA on cytoplasmic Ca(2+) homeostasis, including the protection afforded by oligomycin, could be reproduced by veratridine. KA exposure induced a partial mitochondrial depolarization that was enhanced by oligomycin, indicating ATP synthase reversal. The nonglycolytic substrates pyruvate and lactate were unable to maintain Ca(2+) homeostasis in the presence of KA. In contrast to NMDA, KA exposure did not cause mitochondrial Ca(2+) loading. The data indicate that Na(+) entry via noninactivating AMPA/KA receptors or voltage-activated Na(+) channels compromises mitochondrial function sufficiently to cause ATP synthase reversal. Oligomycin may protect by preventing the consequent mitochondrial drain of cytoplasmic ATP.

Animals↗
PubMed1991

Differences between Lactobacillus casei subsp. casei 2206 and Citrate-Positive Lactococcus lactis subsp. lactis 3022 in the Characteristics of Diacetyl Production.

Lactobacillus casei subsp. casei 2206 exhibited much lower levels of diacetyl reductase activity than CitrLactococcus lactis subsp. lactis 3022 but two-, three-, and more than eightfold-higher levels of diacetyl synthase, lactate dehydrogenase, and NADH oxidase activities, respectively. A requirement for metal ions by the diacetyl synthases in both species was observed. The extracts of strain 2206 but not strain 3022 produced more diacetyl from pyruvate when the reaction for diacetyl synthase was aerated than when it was conducted statically.

Journal Article↗
PubMed1977

Enzyme inactivation by a cellular neutral protease: enzyme specificity, effects of ligands on inactivation, and implications for the regulation of enzyme degradation.

A protease from Tetrahymena pyriformis inactivated eight of nine commercially available enzymes tested, including lactate deyhdrogenase, isocitrate dehydrogenase (TPN-specific), glucose-6 phosphate dehydrogenase, D-amino acid oxidase, fumarase, pyruvate kinase, hexokinase, and citrate synthase. Urate oxidase was not inactivated. Inactivation occurred at neutral pH, was prevented by inhibitors of the protease, and followed first order kinetics. In those cases tested, inactivation was enhanced by mercaptoethanol. Most of the enzyme-inactivating activity was due to a protease of molecular weight 25,000 that eluted from DEAE-Sephadex at 0.3 M KCl. A second protease of this molecular weight, which was not retained by the gel, inactivated only isocitrate dehydrogenase and D-amino acid oxidase. These two proteases could also be distinguished by temperature and inhibitor sensitivity. Two other protease peaks obtained by DEAE-Sephadex chromatography had little or no no enzyme inactivating activity, while another attacked only D-amino acid oxidase. At least six of the enzymes could be protected from proteolytic inactivation by various ligands. Isocitrates dehydrogenase was protected by isocitrate, TPN, or TPNH, glucose-6-dehydrogenase by glucose-6-P or TPN, pyruvate kinase by phosphoenolypyruvate or ADP, hexokinase by glucose, and fumarase by a mixture of fumarate and malate. Lactate dehdrogenase was not protected by either of its substrates of coenzymes. Citrate synthase was probably protected by oxalacetate. Our data suggest that the protease or proteases discussed here may participate in the inactivation or degradation of a least some enzymes in Tetrahymena. Since the inactivation occurs at neutral pH, this process could be regulated by variations in the cellular levels of substrates, coenzymes, or allosteric regulators resulting form changes in growth conditions or growth state. Such a mechanism would permit the selective retention of enzymes of metabolically active pathways.

Animals↗
PubMed2003

Rhodobacter capsulatus 1-deoxy-D-xylulose 5-phosphate synthase: steady-state kinetics and substrate binding.

1-Deoxy-d-xylulose 5-phosphate synthase (DXP synthase) catalyzes the thiamine diphosphate (TPP)-dependent condensation of pyruvate and d-glyceraldehyde 3-phosphate (GAP) to yield DXP in the first step of the methylerythritol phosphate pathway for isoprenoid biosynthesis. Steady-state kinetic constants for DXP synthase calculated from the initial velocities measured at varying concentrations of substrates were as follows: k(cat) = 1.9 +/- 0.1 s(-1), K(m)(GAP) = 0.068 +/- 0.001 mM, and K(m)(pyruvate) = 0.44 +/- 0.05 mM for pyruvate and GAP; k(cat) = 1.7 +/- 0.1 s(-1), K(m)(d-glyceraldehyde) = 33 +/- 3 mM, and K(m)(pyruvate) = 1.9 +/- 0.5 mM for d-glyceraldehyde and pyruvate. beta-Fluoropyruvate was investigated as a dead-end inhibitor for pyruvate. Double-reciprocal plots showed a competitive inhibition pattern with respect to pyruvate and noncompetitive inhibition with respect to GAP/d-glyceraldehyde. (14)CO(2) trapping experiments demonstrated that the binding of both substrates (pyruvate and GAP/d-glyceraldehyde) is required for the formation of a catalytically competent enzyme-substrate complex. These results are consistent with an ordered mechanism for DXP synthase where pyruvate binds before GAP/d-glyceraldehyde.

Bacterial Proteins↗