PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “THIAMINE PYROPHOSPHATE”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,189 records · Page 66Linked to original sources
PubMed2002

The origin of the absorption band induced through the interaction between apotransketolase and thiamin diphosphate.

It has long been known that formation of a catalytically active holotransketolase from the apoenzyme and coenzyme (thiamin diphosphate) is accompanied by the appearance of a new band, in both the absorption and CD spectra. Binding and subsequent conversion of the substrates bring about changes in this band's intensity. The observation of these changes allows the investigator to monitor the coenzyme-to-apoenzyme binding and the conversion of substrates during the transketolase reaction and thus to kinetically characterize its individual steps. The origin of the thiamin diphosphate induced absorption band has been postulated to be resulted from formation of a charge transfer complex or alternatively from an induced conformational transition of the enzyme. The latter brings aromatic amino acid residues into close proximity and generates the absorption. However, X-ray crystallographic and enzyme point mutation experiments cast doubts on both of these hypotheses. Here we show that the binding of thiamin diphosphate to the apotransketolase leads to the conversion of the 4'-amino tautomeric form of its aminopyrimidine ring into the N(1')H-imino tautomeric form. This imino form emerges as a result of the coenzyme's aminopyrymidine ring incorporation into the hydrophobic pocket of the transketolase active center and is stabilized through the interactions with Glu418 and Phe445 residues. The N(1')H-imino tautomeric form of thiamin diphosphate is thought to be the origin of the holotransketolase absorption band induced through the coenzyme binding.

Catalysis↗
PubMed2003

The active site and mechanism of action of recombinant acetohydroxy acid synthase from tobacco.

Acetohydroxy acid synthase (AHAS) is one of several enzymes that require thiamine diphosphate and a divalent cation as essential cofactors. Recently, the three-dimensional structure of the enzyme from yeast has been determined [Pang et al., J. Mol. Biol. 317 (2002) 249-262]. While this structure sheds light on the binding of the cofactors and the reaction mechanism, the interactions between the substrates and the enzyme remain unclear. We have studied the pH dependence of kinetic parameters in order to obtain information about the chemical mechanism in the active site. Data are consistent with a mechanism in which substrate selectively catalyzed to the enzyme with an unprotonated base having a pK of 6.48, and a protonated group having a pK of 8.25 for catalysis. The temperature dependence of kinetic parameters was pH-dependent, and the enthalpies of ionization, DeltaH(ion), calculated from the slope of pK(1) and pK(2) are both pH-independent. The solvent perturbation of kinetic parameters was pH-dependent, and the pK(1) from the acidic side and the pK(2) from the basic side were shifted down 0.4 pH units and shifted up 0.6 units as water was replaced by 15% ethanol, respectively. The data are discussed in terms of the acid-base chemical mechanism.

Acetolactate Synthase↗
PubMed1997

The iron-sulfur centers of the pyruvate:ferredoxin oxidoreductase from Methanosarcina barkeri (Fusaro).

The iron-sulfur clusters of a pyruvate:ferredoxin oxidoreductase isolated from a methanogenic archaeon, Methanosarcina barkeri (Fusaro), have been unambiguously identified for the first time. In agreement with the estimated iron and sulfur contents (Bock and Schonheit, Eur. J. Biochem., 237 (1996) 35-44), the enzyme is shown to contain three [4Fe-4S](2+/1+) clusters, which in the reduced state give a complex EPR spectrum resulting from three distinct centres, magnetically interacting. The catalytic cycle of the enzyme was studied by visible and EPR spectroscopies. A thiamine diphosphate based radical is also an intermediate in the M. barkeri enzyme catalytic cycle. However, under anaerobic conditions, the enzyme or Clostridium pasteurianum ferredoxin iron-sulfur clusters are reduced only in the presence of both substrates, pyruvate and coenzyme A.

Anaerobiosis↗
PubMed1997

Kinetic mechanism of active site non-equivalence in transketolase.

The two-step mechanism of coenzyme (TDP) binding to apotransketolase has been examined by kinetic modeling, and the rate and equilibrium constants for each binding step for two active sites have been determined. The dissociation constants for the primary fast binding step and the forward rate constants for the secondary slow binding step have been shown to be similar for two active sites. The backward rate constants for the secondary binding step are different for two active sites, providing the kinetic mechanism of their non-equivalence in TDP binding.

Binding Sites↗
PubMed1998

Identification of the catalytic glutamate in the E1 component of human pyruvate dehydrogenase.

The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate to acetyl-CoA. The first component (E1) converts pyruvate to bound acetaldehyde using thiamine diphosphate (ThDP) and Mg2+ as cofactors. There is no 3D structure of E1 available but those of other ThDP-dependent enzymes show some similarities including a glutamate residue that assists in ThDP activation. Eukaryotic E1 has an alpha2beta2 structure and the conserved Glu89 of the beta-subunit was identified as a possible catalytic residue by sequence alignment. Human E1 was expressed in Escherichia coli and purified. Mutating Glu89 to glutamine, aspartate and alanine markedly reduces catalytic activity and the affinity for ThDP, consistent with a role as the catalytic glutamate.

Amino Acid Sequence↗
PubMed1998

Activation of thiamine diphosphate in pyruvate decarboxylase from Zymomonas mobilis.

Replacement of tryptophan 392 located in the active site cavity of pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis by methionine or glutamine yields enzymes with smaller catalytic constants of 8.5 s(-1) and 3.6 s(-1) at 4 degrees C, compared to that of the wild-type enzyme (17 s(-1)). The rate constants of the H/D exchange at the C2 of the coenzyme thiamine diphosphate have been determined to be 130 s(-1) for the wild-type enzyme, 56 s(-1) for the methionine and 30 s(-1) for the glutamine mutant, respectively. A group with a pKa of about 5 has been identified to be essential for C2 deprotonation of the enzyme-bound thiamine diphosphate from the pH dependence of the H/D exchange.

Hydrogen-Ion Concentration↗
PubMed1999

Interplay of organic and biological chemistry in understanding coenzyme mechanisms: example of thiamin diphosphate-dependent decarboxylations of 2-oxo acids.

With the publication of the three-dimensional structures of several thiamin diphosphate-dependent enzymes, the chemical mechanism of their non-oxidative and oxidative decarboxylation reactions is better understood. Chemical models for these reactions serve a useful purpose to help evaluate the additional catalytic rate acceleration provided by the protein component. The ability to generate, and spectroscopically observe, the two key zwitterionic intermediates invoked in such reactions allowed progress to be made in elucidating the rates and mechanisms of the elementary steps leading to and from these intermediates. The need remains to develop chemical models, which accurately reflect the enzyme-bound conformation of this coenzyme.

Acetoin Dehydrogenase↗
PubMed1990

Determination of water-soluble vitamins in blood and plasma by coupled-column liquid chromatography.

A method is described for the simultaneous determination of riboflavin and the coenzymes related to pyridoxine and thiamine. Pyridoxal-5'-phosphate (PLP) bound to proteins as a Schiff base was liberated and stabilized by reaction with semicarbazide. Blood or plasma proteins were precipitated with acid and the supernatant formed was injected into a liquid chromatographic system consisting of one precolumn and two analytical reversed-phase columns. Riboflavin was detected by its native fluorescence with a separate detector while post-column reactions based on the fluorescence of PLP semicarbazone in alkaline solution and the oxidation of thiamine to thiochrome were combined in the same reaction detector. Chromatographic separation was achieved within 7 min and normal endogenous levels were quantified with a precision of 2-5% (relative standard deviation).

Chromatography, Liquid↗
PubMed1998

Thiamine and its derivatives inhibit delayed rectifier potassium channels of rat cultured cortical neurons.

We examined the effects of thiamine and its derivatives on voltage-gated ion channels of neuronal cells isolated from fetal forebrain cortex and cultured for 6-14 days. Under the whole-cell voltage clamp, thiamine tetrahydrofurfuryl disulfide (TTFD), a membrane-permeable derivative of thiamine, inhibited the delayed rectifier K+ current (IK) in a concentration-dependent manner (10(-4)-10(-3) M). The IK-suppressing effect was also observed by internal perfusion with 1 mM thiamine, but not by the external application of thiamine, indicating the poor permeability of thiamine through the cell membrane. However, thiamine which was applied directly to the intracellular side of patch membranes in the inside-out configuration failed to decrease the open probability of the single IK channel. In contrast, thiamine diphosphate decreased both the open probability and the open-time of the channel without changing the single channel conductance. These results suggest that phosphorylated thiamine can function as an endogenous K+ channel blocker in neuronal cells. TTFD, when applied extracellularly at a concentration of 1 mM, prolonged the action potential (AP) duration of neurons (172.8 +/- 6.6%) without changing the resting membrane potential or AP amplitude, while the same concentration of thiamine did not influence any parameters of the AP, implying that TTFD may cause the potentiation of neuronal AP through the inhibition of IK.

Animals↗