丙酮酸脱羧酶及其应用研究

丙酮酸脱羧酶(pyruvate decarboxylase,PDC),EC4.1.1.1,是一种胞内酶,是焦磷酸硫胺素(thiamine pyrophosphate,ThPP)依赖性的非氧化酶,是由辅酶ThPP、Mg2+和蛋白质构成的全酶,在辅助因子焦磷酸硫胺素和Mg2+参与下作用于丙酮酸而产生乙醛和CO2。PDC是丙酮酸合成乙醇的关键酶。它广泛存在于酵母菌、霉菌、细菌和植物等多种生物体中,不同来源的丙酮酸脱羧酶的结构、相对分子质量、酶学性质等均不尽相同。该文综述了丙酮酸脱羧酶生物学性质及其应用前景。     

Identification and reconstitution of the yeast mitochondrial transporter for thiamine pyrophosphate

The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 15 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates and cofactors across the inner membrane. Here the identification of the mitochondrial carrier for the essential cofactor thiamine pyrophosphate (ThPP) is described. The protein has been overexpressed in bacteria, reconstituted into phospholipid vesicles and identified by its transport properties. In confirmation of its identity, cells lacking the gene for this carrier had reduced levels of ThPP in their mitochondria, and decreased activity of acetolactate synthase, a ThPP-requiring enzyme found in the organellar matrix. They also required thiamine for growth on fermentative carbon sources.     

Purification and characterization of pyruvate dehydrogenase complex from borccoli floral buds.

The pyruvate dehydrogenase complex (PDC) was purified from Brassica oleracea var. italica floral buds to a specific activity of approximately 6 μmol of NADH formed/min/ mg of protein. The PDC had cofactor requirements for NAD⁺, thiamine pyrophosphate, coenzyme A, and a divalent cation (Mg²⁺, Ca²⁺, or Mn²⁺). The enzyme catalyzed the oxidative decarboxylation of pyruvate at a rate threefold faster than 2-oxobutyrate but was inactive toward 2-oxoglutarate. The PDC was competively inhibited by acetyl-CoA against CoA and NADH against NAD⁺. The enzyme was shown to be more sensitive to regulation by NADH than acetyl-CoA.     

Thiamine repression and pyruvate decarboxylase autoregulation independently control the expression of the Saccharomyces cerevisiae PDC5 gene.

The Saccharomyces cerevisiae gene PDC5 encodes the minor isoform of pyruvate decarboxylase (Pdc). In this work we show that expression of PDC5 but not that of PDC1, which encodes the major isoform, is repressed by thiamine. Hence, under thiamine limitation both PDC1 and PDC5 are expressed. PDC5 also becomes strongly expressed in a pdc1delta mutant. Two-dimensional gel electrophoresis of whole protein extracts shows that thiamine limitation stimulates the production of THI gene products and of Pdc5p. Deletion of PDC1 only stimulates production of Pdc5p. We conclude that the stimulation of PDC5 expression in a pdc1delta mutant is not due to a response to thiamine limitation.     

Thiamine diphosphate pool and its biosynthesis in the liver in galactosamine hepatitis

The hepatitis-like changes were induced in the liver of albino female rats weighing 120-150 g and fed on the appropriate vivarium diet by single parenteral administration of hydrochloride galactosamine in a dose of 0.9 or 1.8 mmol per 1 kg of body weight. The thiamine diphosphate level in the cytosol fraction of the liver decreased 24 h after the preparation administration, the same in blood but with the higher dose used. The activity of pyruvate dehydrogenase, a thiamine diphosphate dependent enzyme, decreased similarly. The cytosol transketolase activity lowered by 38-39%. The coenzyme biosynthesis disturbance due to a fall by 49-58% in the thiamine pyrophosphatase activity is considered to be responsible for hydrochloride galactosamine-induced decrease in the thiamine diphosphate pool. Specificity of the thiamine diphosphate pool disturbance and discoordination of thiamine diphosphate dependent enzymes in the liver are observed under administration of hydrochloride galactosamine.     

31P NMR investigations on free and enzyme bound thiamine pyrophosphate

Pyruvate decarboxylase (PDC) contains thiamine pyrophosphate (TPP) and Mg2+ as cofactors. 31P NMR studies with PDC in the presence of added Mn2+ reveal the pyrophosphate moiety of TPP to be a nonaccessible area for the external Mn2+ and thus proving the Mg-P-complex (taking part in the binding of the coenzyme to the protein) to be a nonaccessible area for the medium. Glyoxylic acid, acting as an inhibitor of PDC by forming a noncleavable bond with the catalytic center of TPP causes a steric immobilization of the coenzyme indicated by a line broadening of the pyrophosphate moiety.     

Regulation of pea mitochondrial pyruvate dehydrogenase complex activity: inhibition of ATP-dependent inactivation

In contrast to the pyruvate dehydrogenase complex (PDC) from animal mitochondria, our in situ and in vitro studies indicate that the ATP:ADP ratio has little or no effect in regulating the mitochondrial pyruvate dehydrogenase complex from green pea seedlings. Pyruvate was a competitive inhibitor of ATP-dependent inactivation (Ki = 59 microM), while the PDC had a Km for pyruvate of microM. Thiamine pyrophosphate, the coenzyme for the pyruvate dehydrogenase (PDH) component of the complex, did not inhibit ATP-dependent inactivation when used alone but it enhanced inhibition by pyruvate. As such, thiamine pyrophosphate was a competitive inhibitor (Ki = 130 nM) of ATP-dependent inactivation. A model is proposed for the pyruvate plus thiamine pyrophosphate inhibition of ATP-dependent inactivation of the pyruvate dehydrogenase complex in which pyruvate exerts its inhibition of inactivation by altering or protecting the protein substrate from phosphorylation and not by directly inhibiting PDH kinase.     

A rapid and sensitive radiometric assay procedure for thiamine pyrophosphokinase activity using anion-exchange paper discs

A radiochemical method for the direct measurement of thiamine pyrophosphokinase (ATP: thiamine pyrophosphotransferase, EC 2.7.6.2) activity was described earlier (1,2). It avoided the difficulties associated with assay systems based on the coenzyme nature of thiamine pyrophosphate in TPP-dependent¹ enzyme reactions using apopyruvate decarboxylase (3) (2-oxoacid carboxylase, EC 4.1.1.1) or apotransketolase (4) (sedoheptulose-7-phosphate: d-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1). Since the chromatographic isolation of TPP is time-consuming, a procedure for the rapid determination of thiamine pyrophosphokinase activity was desirable.The simplified method described here takes advantage of the anionic character of TPP. The assay is carried out with [¹⁴C]thiamine as substrate. After incubation with the enzyme in the presence of Mg²⁺-ATP, the reaction mixture is applied to a DEAE-cellulose paper disc. The disc is extensively washed with sodium acetate resulting in the quantitative elution of [¹⁴C]thiamine and partial retention of [¹⁴C]TPP. This is quantitatively measured using the liquid scintillation counting technique.A similar procedure has been described for the determination of glycerol kinase (ATP: glycerol phosphotransferase, EC 2.7.1.30) and hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) activities (5).     

Deciphering regulatory variation of THI genes in alcoholic fermentation indicate an impact of Thi3p on PDC1 expression

Background

Thiamine availability is involved in glycolytic flux and fermentation efficiency. A deficiency of this vitamin may be responsible for sluggish fermentations in wine making. Therefore, both thiamine uptake and de novo synthesis could have key roles in fermentation processes. Thiamine biosynthesis is regulated in response to thiamine availability and is coordinated by the thiamine sensor Thi3p, which activates Pdc2p and Thi2p. We used a genetic approach to identify quantitative trait loci (QTLs) in wine yeast and we discovered that a set of thiamine genes displayed expression-QTL on a common locus, which contains the thiamine regulator THI3.

Results

We deciphered here the source of these regulatory variations of the THI and PDC genes. We showed that alteration of THI3 results in reduced expression of the genes involved in thiamine biosynthesis (THI11/12/13 and THI74) and increased expression of the pyruvate decarboxylase gene PDC1. Functional analysis of the allelic effect of THI3 confirmed the control of the THI and PDC1 genes. We observed, however, only a small effect of the THI3 on fermentation kinetics. We demonstrated that the expression levels of several THI genes are correlated with fermentation rate, suggesting that decarboxylation activity could drive gene expression through a modulation of thiamine content. Our data also reveals a new role of Thi3p in the regulation of the main pyruvate decarboxylase gene, PDC1.

Conclusions

This highlights a switch from PDC1 to PDC5 gene expression during thiamine deficiency, which may improve the thiamine affinity or conservation during the enzymatic reaction. In addition, we observed that the lab allele of THI3 and of the thiamin transporter THI7 have diverged from the original alleles, consistent with an adaptation of lab strains to rich media containing an excess of thiamine.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1085) contains supplementary material, which is available to authorized users.     

Regulation of steady state pyruvate dehydrogenase complex activity in plant mitochondria : reactivation constraints

The requirements for reactivation (dephosphorylation) of the pea (Pisum sativum L.) leaf mitochondrial pyruvate dehydrogenase complex (PDC) were studied in terms of magnesium and ATP effects with intact and permeabilized mitochondria. The requirement for high concentrations of magnesium for reactivation previously reported with partially purified PDC is shown to affect inactivation rather than reactivation. The observed rate of inactivation catalyzed by pyruvate dehydrogenase (PDH) kinase is always greater than the reactivation rate catalyzed by PDH-P phosphatase. Thus, reactivation would only occur if ATP becomes limiting. However, pyruvate which is a potent inhibitor of inactivation in the presence of thiamine pyrophosphate, results in increased PDC activity. Analysis of the dynamics of the phosphorylation-dephosphorylation cycle indicated that the covalent modification was under steady state control. The steady state activity of PDC was increased by addition of pyruvate. PDH kinase activity increased threefold during storage of mitochondria suggesting that there may be an unknown level of regulation exerted on the enzyme complex.     

Inhibition of transketolase by hexacyanoferrate(III)

The effect of hexacyanoferrate(III) on the catalytic activity of transketolase has been studied. This oxidant inactivates only one of two active sites of the enzyme, the one with a higher affinity to the coenzyme (thiamine diphosphate). The second active site does not lose its catalytic activity. These observations indicate that the active sites of holotransketolase, being indiscernible by data of X-ray analysis, exhibit functional nonequivalence.     

The role of bound thiamine pyrophosphate in the synthesis of thiamine triphosphate in rat liver.

Thiamine pyrophosphate-ATP phosphoryltransferase, the enzyme that catalyzes the synthesis of thiamine triphosphate, has been found in the supernatant fraction of rat liver. The substrate for the enzyme is endogenous, bound thiamine pyrophosphate, since the addition of exogenous thiamine pyrophosphate had no effect. Thus, when a rat liver supernatant was incubated with gamma-labelled [32P]ATP, thiamine [32P]triphosphate was formed whereas the incubation of thiamine [32P]pyrophosphate with ATP did not produce thiamine [32P]triphosphate. The endogenous thiamine pyrophosphate was found to be bound to a high molecular weight protein which comes out in the void volume of Sephadex G-75, and is not dialyzable. The activity that catalyzes the formation of thiamine triphosphate has an optimum pH between 6 and 6.5, a linear time course of thiamine triphosphate synthesis up to 30 min, and is not affected by Ca2+, cyclic GMP and sulfhydryl reagents.     

The nature of binding between the coenzyme and protein in transketolase from the swine liver

An analysis of a proteolytic hydrolysate of pig liver transketolase by thin-layer chromatography revealed the presence of a coenzyme-containing material which differed from free thiamine pyrophosphate in chromatographic behaviour. This coenzyme-containing material is distinct from the free coenzyme in terms of other properties as well, e.g., stability, pH dependence of thiochrome fluorescence, etc. It was demonstrated that incubation of enzyme preparations possessing a high specific activity (on the average, 2 E/mg) in acidic acetate buffer caused no or little detachment of the coenzyme, mainly in the composition of the heterogeneous material which, at least partly, was not represented by thiamine pyrophosphate.     

Purification of a branched-chain keto acid dehydrogenase from Pseudomonas putida.

We purified branched-chain keto acid dehydrogenase to a specific activity of 10 mumol/min per mg of protein from Pseudomonas putida grown on valine. The purified enzyme was active with 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate in a ratio of 1.0:0.8:0.7 but showed no activity with either pyruvate or 2-ketoglutarate. There were four polypeptides in the purified enzyme (molecular weights, 49,000, 46,000, 39,000, and 37,000). The purified enzyme was deficient in the specific lipoamide dehydrogenase produced during growth on valine (molecular weight, 49,000). Branched-chain keto acid dehydrogenase required L-valine, oxidized nicotinamide adenine dinucleotide, coenzyme A, thiamine pyrophosphate, and magnesium chloride. A partially purified preparation catalyzed the oxidation of 2-keto-[1-14C]isovalerate to [14C]carbon dioxide, isobutyryl-coenzyme A, and reduced nicotinamide adenine dinucleotide in equimolar amounts. Both the Km and the Vmax for 2-ketoisovalerate were affected by the addition of L-valine to the assay mixture. However, only the Vmax values for oxidized nicotinamide adenine dinucleotide and coenzyme A were affected when L-valine was present. This suggested that valine acted by affecting the binding of branched-chain keto acids to subunit E1 of the complex.     

Regulation of the phosphorylation of mitochondrial pyruvate dehydrogenase complex in situ: effects of respiratory substrates and calcium

The activity of the pyruvate dehydrogenase complex (PDC), as controlled by reversible phosphorylation, was studied in situ with mitochondria oxidizing dfifferent substrates. PDCs from both plant and animal tissues were inactivated when pyruvate became limiting. The PDC did not inactivate in the presence of saturating levels of pyruvate. Calcium stimulated reactivation of PDC in chicken heart but not pea (Pisum sativum L.) leaf mitochondria. With pea leaf mitochondria oxidizing malate, inactivation of PDC was pH dependent corresponding to the production of pyruvate via malic enzyme. When pea leaf mitochondria oxidized succinate or glycine, PDC was inactivated. This inactivation was reversed by the addition of pyruvate. Reactivation by pyruvate was enhanced by the addition of thiamine pyrophosphate, as previously observed with nonrespiring mitochondria. These results indicate a major role for pyruvate in regulating the covalent modification of the PDC.     

Characteristics of thiamine thiazolone diphosphate-induced inhibition of a pyruvate dehydrogenase complex in vitro and in intact mitochondria

Thiamine thiazolone diphosphate (TTPP) was capable of penetrating through the mitochondrial membrane and of inhibiting the pyruvate dehydrogenase complex (PDC) in intact mitochondria. TTPP depressed the activity of mammalian PDC in a mixed manner (Ki = 5.10(-8) M) and yeast pyruvate decarboxylase (Ki = 5.10(-6) M) via a competitive mechanism with respect to thiamine diphosphate. It was shown that decarboxylation of pyruvate in intact and disrupted mitochondria of rat liver and brain is less inhibited by TTPP than the overall activity of PDC determined by the formation of acetyl-CoA. It was assumed that TTPP as a transition state analog participates only in oxidative reactions (but not in simple decarboxylation of pyruvate).     

Effect of thiamine and its metabolites on aspartate and alanine aminotransferase activity in the body of white rats and in donor blood

ThPP and thiochrome, being thiamine metabolites, are inhibitors of blood transaminase. Their action is evidently realized on the level of competition with PALP when these compounds attach to apotransaminases.     

Thiamine preserves mitochondrial function in a rat model of traumatic brain injury,preventing inactivation of the 2-oxoglutarate dehydrogenase complex

Background and purpose

Based on the fact that traumatic brain injury is associated with mitochondrial dysfunction we aimed at localization of mitochondrial defect and attempted to correct it by thiamine.

Thiamine binding and metabolism in germinating seeds of selected cereals and legumes.

The basic characteristics of thiamine metabolism in germinating seeds of maize (Zea mays), oat (Avena sativa), faba bean (Vicia faba) and garden pea (Pisum sativum) are presented with a special emphasis of a possible thiamine storage function of seed thiamine-binding proteins (TBPs). Seeds were germinated for 6 d in the dark. Thiamine-binding activity in seeds decreased during germination by 50% in cereals and by 30% in legumes. The degradation of TBPs was also detected by polyacrylamide gel electrophoresis. The total thiamine content decreased rapidly to 20-40% of the initial value in cereal seeds during first 3 d of germination while in legume seeds thiamine content started changing from the fourth day and dropped by 50% at the sixth day. A composite pattern was found for the changes in thiamine pyrophosphate (TPP) contribution to total thiamine during seed germination. A peak of the coenzyme percentage was usually detected at the second day of germination. Another gain of TPP was often seen toward the sixth day of germination. The activity of thiamine pyrophosphokinase (EC 2.7.6.2) was high in resting legume seeds and did not significantly change during germination. In contrast, the low activity of this thiamine-activating enzyme in cereal seeds progressively increased during germination. Thiamine phosphate synthase (EC 2.5.1.3) was also detected in seeds and was shown to contribute significantly to the balance of thiamine compounds during seed germination.