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Preparations of a highly purified pyruvate dehydrogenase complex (PDC) from human heart contain endogenous thiamine pyrophosphate (TPP) in an amount accounting for about 10% of the maximum activity. At pH values of 7.5 and 8.0, the effective Michaelis constants with respect to exogenous TPP for the PDC apoenzyme form were 0.22 microM and 1.8 microM, respectively.
The development of vitamin B1 (thiamine) deficiency was investigated in 17 obese patients in the course of a 1000 calory diet or during total fasting for a fortnight. Compared with normal persons no changes of concentrations of total thiamine, TPP, and pyruvate in the blood; thiamine excretion in the urine, or activiation coefficient of the eryhrocyte transketolase (alphaETK) were observed during the reducing diet. However, during a fortnight's total fasting all values decreased significantly to those of thiamine deficiency. The thiamine pyrophosphate (TPP) levels in the blood dropped from the 1st to the 14th day, whereas the alphaETK rose accordingly and the pyruvate levels showed a delayed rise. Total thiamine content of the blood and thiamine excretion in the urine only showed significantly different values when comparing the 1st and the 14th day. There was no dependency of thiamine excretion on urinary output. Clinical symptoms of thiamine deficiency could be demonstrated in no case.
Administration of the vitamin-coenzyme complex containing thiamin pyrophosphate:lipoate:4-phosphopantothenate:nicotinate:flavinad enine mononucleotide (0.2:3:5:8:1) led to increased utilization of alpha-ketoglutarate in nervous tissue via alpha-ketoglutarate dehydrogenase and GABA-transaminase pathways. In these systems content of glutamate and GABA was decreased, suggesting their elevated consumption as energy-providing substrates. If 4-phosphopantothenate was substituted by CoASH and nicotinate--by NAD+, increased decarboxylation of 5(-14)C-alpha-ketoglutarate, 1(-14)C-GABA and 4(-14)C-Asp was detected in brain homogenates in vitro. The neurometabolic effects were accompanied by pronounced antihypoxic effects as shown by the test "hypoxy of closed space".
Oxythiamine is an antivitamin derivative of thiamine that after phosphorylation to oxythiamine pyro phosphate can bind to the active centres of thiamine-dependent enzymes. In the present study, the effect of oxythiamine on the viability of Saccharomyces cerevisiae and the activity of thiamine pyrophosphate dependent enzymes in yeast cells has been investigated. We observed a decrease in pyruvate decarboxylase specific activity on both a control and an oxythiamine medium after the first 6 h of culture. The cytosolic enzymes transketolase and pyruvate decarboxylase decreased their specific activity in the presence of oxythiamine but only during the beginning of the cultivation. However, after 12 h of cultivation, oxythiamine-treated cells showed higher specific activity of cytosolic enzymes. More over, it was established by SDS-PAGE that the high specific activity of pyruvate decarboxylase was followed by an increase in the amount of the enzyme protein. In contrast, the mitochondrial enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, were inhibited by oxythiamine during the entire experiment. Our results suggest that the observed strong decrease in growth rate and viability of yeast on medium with oxythiamine may be due to stronger inhibition of mitochondrial pyruvate dehydrogenase than of cytosolic enzymes.
Inhibitory effects of 4'-oxythiamine pyrophosphate (OTPP) and tetrahydrothiamine pyrophosphate (ThTPP) on the purified bison heart pyruvate dehydrogenase complex (PDC) semisaturated with endogenous thiamine pyrophosphate (TPP) and 2-oxoglutarate dehydrogenase complex (OGDC) saturated about 85% with endogenous TPP, were studied. It has been established that the thiamine derivatives strongly inhibit not only the PDC apoenzyme moiety, but also the PDC holoenzyme moiety. The apparent I50 values for the holoenzyme were 0.006 microM and 0.046 microM for OTPP and ThTPP, respectively. The inhibition of the PDC is reversible. After removal of the anticoenzyme analogues by gel filtration the endogenous TPP within the PDC is retained as it is evidenced by complete recovery of the enzyme activity without added TPP. In contrast with the PDC, OGDC holoenzyme form is weakly inhibited by the anticoenzyme analogues.
1. The binding kinetics for [35S]thiamine pyrophosphate to transketolase and the dependency of transketolase on divalent cations for activity were investigated. 2. With Scatchard analysis, dissociation constant (Kd) and n value were calculated to be 0.2 x 10(-6) M and 0.66 respectively. 3. The activity of the reconstituted enzyme increased in the order of Co2+ less than Mn2+ less than Ca2+ less than Mg2+. The native transketolase contained Mg2+ in its molecular structure.
The mode of interaction between Escherichia coli pyruvate oxidase and its cofactor, thiamin pyrophosphate (TPP), was studied with the aid of arginine-directed reagents. The enzyme is rapidly inactivated by either phenylglyoxal or 2,3-butanedione, with the cofactor, TPP, offering partial protection against these reagents. The inactivation by phenylglyoxal was found to be reversible. Experiments with [7-14C]phenylglyoxal showed that while several arginine residues react with this reagent, TPP can prevent the labeling of one such residue. Furthermore, inactivation by 2,3-butanedione is attended by at least a 100-fold decrease in affinity of the enzyme for TPP. These results suggest a direct role for arginine in the binding of the cofactor.
The human pyruvate dehydrogenase complex (PDHC) catalyzes the thiamine-dependent decarboxylation of pyruvate. Thiamine treatment is very effective for some patients with PDHC deficiency. Among these patients, five mutations of the pyruvate dehydrogenase (E1)alpha subunit have been reported previously: H44R, R88S, G89S, R263G, and V389fs. All five mutations are in a region outside the thiamine pyrophosphate (TPP)-binding region of the E1alpha subunit. We report the biochemical and molecular analysis of two patients with clinically thiamine-responsive lactic acidemia. The PDHC activity was assayed using two different concentrations of TPP. These two patients displayed very low PDHC activity in the presence of a low (1 x 10(-4) mM) TPP concentration, but their PDHC activity significantly increased at a high (0.4 mM) TPP concentration. Therefore, the PDHC deficiency in these two patients was due to a decreased affinity of PDHC for TPP. Treatment of both patients with thiamine resulted in a reduction in the serum lactate concentration and clinical improvement, suggesting that these two patients have a thiamine-responsive PDHC deficiency. The DNA sequence of these two male patients' X-linked E1alpha subunit revealed a point mutation (F205L and L216F) within the TPP-binding region in exon 7.
The kinetic behavior of pig heart pyruvate dehydrogenase complex (PDC) containing bound endogenous thiamin pyrophosphate (TPP) was affected by exogenous TPP. In the absence of exogenous TPP, a lag phase of the PDC reaction was observed. TPP added to the PDC reaction medium containing Mg2+ led to a disappearance of the lag phase, inducing strong reduction of the Km value for pyruvate (from 76.7 to 19.0 microM) but a more moderate decrease of Km for CoA (from 12.2 to 4.3 microM) and Km for NAD+ (from 70.2 to 33.6 microM), with no considerable change in the maximum reaction rate. Likewise, thiamin monophosphate (TMP) decreased the Km value of PDC for pyruvate, but to a lesser extent (from 76.7 to 57.9 microM) than TPP. At the unsaturating level of pyruvate, the A50 values for TPP and TMP were 0.2 microM and 0.3 mM, respectively. This could mean that the effect of TPP on PDC was more specific. In addition, exogenous TPP changed the UV spectrum and lowered the fluorescence emission of the PDC containing bound endogenous TPP in its active sites. The data obtained suggest that TPP plays, in addition to its catalytic function, the important role of positive regulatory effector of pig heart PDC.
A sensitive method for the specific measurement of thiamin pyrophosphate (TPP) has been developed using the apoenzyme recombination concept. Yeast pyruvic decarboxylase apoenzyme can be reconstituted by the addition of TPP or samples containing TPP, yielding the holoenzyme with activity proportionate to the amount of TPP added. Using this technique, reaction mixtures containing 0.2 to 1.5 ng TPP can be assayed. Normal human erythrocyte TPP ranges from 50 to 150 ng per ml packed cells. When rats are fed a thiamin deficient diet, the erythrocyte TPP level falls more rapidly than the erythrocyte transketolase activity. After 8 days, the level of TPP in the erythrocytes of deficient animals was 10% of the level in pair-fed controls. At this time, however, there was no appreciable decrease in their respective transketolase activities. The level of TPP in the liver also is decreased drastically after 8 days. Therefore it appears that erytyrocyte and liver TPP stores have begun to be depleted and suggest that erythrocyte TPP levels are a more sensitive indicator of thiamin status.
Thiamine pyrophosphate and sodium dichloroacetate, substances which promote the aerobic metabolism of pyruvate, reduce the increase in lactate output induced by phenformin (100 mg/l) on the isolated and perfused rat pancreas. Both substances provoke a decrease in insulin secretion produced by phenformin.
Earlier it was noted that purified pyruvate dehydrogenase complex (PDC) produced by "Sigma" usually contains almost saturating amounts of thiamine pyrophosphate (ThPP). In this communication we present the observation that the endogenous ThPP coupled to PDC is dephosphorylated while staying at -10 degrees C, because in the enzyme preparation thiamine monophosphate and un-phosphorylated thiamine appear (HPLC determination). Under the same conditions exogenous ThPP is not dephosphorylated despite contact with the PDC preparation. This may suggest that interactions of some active groups of the enzyme with molecules of endogenous ThPP leads to break-up of the phosphoesters bonds, and destruction of the coenzyme. Decrease of PDC activity during storage is not in proportion with the degree of ThPP dephosphorylation. However the observed instability of PDC activity may be a consequence of the spontaneous process of its coenzyme autodestruction.
Riboswitches are highly structured RNA elements that control gene expression by binding directly to small metabolite molecules. Remarkably, many of these metabolites contain negatively charged phosphate groups that contribute significantly to the binding affinity. An example is the thiamine pyrophosphate-sensing riboswitch in the 5'-untranslated region of the E. coli thiM mRNA. This riboswitch binds, in order of decreasing affinity, to thiamine pyrophosphate (TPP), thiamine monophosphate (TMP), and thiamine, which contain two, one, and no phosphate groups, respectively. We examined the binding of TPP and TMP to this riboswitch by using (31)P NMR spectroscopy. Chemical-shift changes were observed for the alpha- and beta-phosphate group of TPP and the phosphate group of TMP upon RNA binding; this indicates that they are in close contact with the RNA. Titration experiments with paramagnetic Mn(2+) ions revealed strong line-broadening effects for both (31)P signals of the bound TPP; this indicates a Mg(2+) binding site in close proximity and suggests that the phosphate group(s) of the ligand is/are recognized in a magnesium ion-mediated manner by the RNA.
The contractile activity of the guinea pig taenia coli longitudinal muscle was studied as affected by exogenic thiamine, thiamine pyrophosphate and FAD. The mentioned substances in a concentration of 1 . 10(-3) M relax the smooth-muscular cells. Against a background of apamine FAD causes a contraction of taenia coli, which is not observed with application of thiaminepyrophosphate. Evidently, there are two, types of receptors sensitive to FAD and one sensitive to vitamin B1 on the membrane of the smooth-muscular cell.
While most of the proteins required for the biosynthesis of thiamin pyrophosphate have been known for more than a decade, the reconstitution of this biosynthesis in a defined biochemical system has been difficult due to the novelty of the chemistry involved. Here we demonstrate the first successful enzymatic synthesis of the thiazole moiety of thiamin from glycine, cysteine, and deoxy-D-xylulose-5-phosphate using overexpressed Bacillus subtilis ThiF, ThiS, ThiO, ThiG, and a NifS-like protein. This has facilitated the identification of the biochemical function of each of the proteins involved: ThiF catalyzes the adenylation of ThiS; NifS catalyzes the transfer of sulfur from cysteine to the acyl adenylate of ThiS; ThiO catalyzes the oxidation of glycine to the corresponding imine; and ThiG catalyzes the formation of the thiazole phosphate ring. The complex oxidative cyclization reaction involved in the biosynthesis of the thiamin thiazole has been greatly simplified by replacing ThiF, ThiS, ThiO, and NifS with defined biosynthetic intermediates in a reaction where ThiG is the only required enzyme.
Highly purified preparations of the pyruvate dehydrogenase complex from bovine adrenals partially contain strongly bound thiamine pyrophosphate (TPP) which provides to 35% of the maximal activity measured under saturation with the exogenous TPP. The dependence of the complex-catalyzed reaction rate on the TPP concentration is described by Michaelis-Menten equation. The apparent value of Km for TPP without Mg2+ is 2.3 mumol. Magnesium ions reduce Km to 1.1 mumol. The constant of the TPP with the enzyme association rate calculated by the lag-period without Mg2+ is 3043 mol-1 s-1 in the presence of Mg2+ it is 9090 mol-1 . s-1. Phosphorus ethers of oxy- and tetrahydrothiamine produce a competitive type inhibition on the pyruvate dehydrogenase complex with respect to TPP. Oxythiamine pyrophosphate (Ki--0,07 microM) and tetrahydrothiamine pyrophosphate (Ki--0,1 microM) possess the highest inhibitory action.
The purified 2-oxoglutarate dehydrogenase complex (OGDC) from the European bison heart was near saturated with endogenous bound thiamine pyrophosphate (TPP). Exogenous TPP added to the full OGDC reaction medium decreased S0.5 for 2-oxoglutarate approximately 2.6-fold without any notable change in the maximum reaction rate. The TPP effect was observed in the presence of 1 mM ADP which alone is a strong positive allosteric effector of OGDC. At an unsaturating 2-oxoglutarate concentration the A50 value for TPP was approximately 0.05 mM. The ADP-like action of exogenous TPP was also found in the 2-oxoglutarate dehydrogenase (E1) reaction, determined in the presence of 2,6-dichlorophenoloindophenol as an electron acceptor.
We identified a new Y243S mutation in the X-linked E1 alpha-PDH gene in a patient with pyruvate dehydrogenase complex (PDHc) deficiency. The activity in cultured fibroblasts was very low even in the presence of high thiamine pyrophosphate (TPP) concentrations, indicating that the defect could be due to decreased affinity of PDHc for TPP.