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PubMedDate not supplied

Long-term therapy with cytochrome c, flavin mononucleotide and thiamine diphosphate for a patient with Kearns-Sayre syndrome.

Cardiocrome, containing cytochrome c, flavin mononucleotide and thiamine diphosphate, was administered intravenously for 22 months to a patient with Kearns-Sayre syndrome. This combined therapy alleviated the patient's easy fatigability, motor disability, corneal edema and chilblains, but was not effective for his ophthalmoplegia, blepharoptosis or hearing loss. Truncal ataxia, dysphagia and an atrioventricular block appeared even with this therapy. Although the abnormal distribution of cerebral blood flow demonstrated by single photon emission computed tomography was improved, serial cranial magnetic resonance imaging and electrophysiological examination revealed progressive changes. In conclusion, this therapy was favorably effective for impaired skeletal muscle function and corneal edema, but not for ocular movements, central nervous system symptoms or cardiac conduction abnormalities, because irreversible degeneration had probably occurred in these organs.

Cytochrome c Group↗
PubMed1993

A thiamin diphosphate binding fold revealed by comparison of the crystal structures of transketolase, pyruvate oxidase and pyruvate decarboxylase.

BACKGROUND: The crystal structures of three thiamin diphosphate-dependent enzymes that catalyze distinct reactions in basic metabolic pathways are known. These enzymes--transketolase, pyruvate oxidase and pyruvate decarboxylase--also require metal ions such as Ca2+ and Mg2+ as cofactors and have little overall sequence similarity. Here, the crystal structures of these three enzymes are compared. RESULTS: The three enzymes share a similar pattern of binding of thiamin diphosphate and the metal ion cofactors. The enzymes function as multisubunit proteins, with each polypeptide chain folded into three alpha/beta domains. Two of these domains are involved in binding of the thiamin diphosphate and the metal ion. These domains have the same topology of six parallel beta-strands and surrounding alpha-helices. The thiamin diphosphate is bound in a cleft, formed by two domains from two different subunits. Only a few residues are conserved in all three enzymes and these are responsible for proper binding of the cofactors. CONCLUSIONS: Despite considerable differences in quaternary structure and lack of overall sequence homology, thiamin diphosphate binds to the three enzymes in a very similar fashion, and a general thiamin-binding fold can be revealed.

Amino Acid Sequence↗
PubMed2004

The role of cysteine 160 in thiamine diphosphate binding of the Calvin-Benson-Bassham cycle transketolase of Rhodobacter sphaeroides.

The transketolase gene (cbbT) that encodes the Calvin-Benson-Bassham pathway transketolase (CbbT) of Rhodobacter sphaeroides was overexpressed in Escherichia coli and the recombinant protein purified to homogeneity. Like other transketolases, R. sphaeroides CbbT was found to be inactivated in the presence of oxygen. At its optimal pH of 7.8, CbbT displays a specific activity of 37 U/mg, a KR5P of 949 microM, a KXu5P of 11 microM, and a KThDP of 1.8 microM. Cysteine 160, equivalent to Cys159 of the yeast enzyme, is found within the active site and is loosely conserved amongst several sources of transketolase. To investigate the role of cysteine 160 found in the active site of R. sphaeroides CbbT, this residue was targeted for mutagenesis. Cys160 was changed to alanine, serine, aspartate, and glutamate. To compare the effect of these mutations on ThDP binding, spectral techniques were employed in addition to analysis by enzymatic activity. Fluorescence quenching was used to measure both equilibrium binding constants as well as first order rates of binding. The results of these studies indicated that Cys160 played an important and substantial role in cofactor binding, revealing the importance of this loosely conserved residue. In addition, the Cys160 mutants did not appear to alter oxygen-mediated inactivation.

Air↗
PubMed2005

Experimental observation of thiamin diphosphate-bound intermediates on enzymes and mechanistic information derived from these observations.

Thiamin diphosphate (ThDP), the vitamin B1 coenzyme, is an excellent representative of coenzymes, which carry out electrophilic catalysis by forming a covalent complex with their substrates. The function of ThDP is to greatly increase the acidity of two carbon acids by stabilizing their conjugate bases, the ylide/C2-carbanion of the thiazolium ring and the C2alpha-carbanion (or enamine) once the substrate binds to ThDP. In recent years, several ThDP-bound intermediates on such pathways have been characterized by both solution and solid-state (X-ray) methods. Prominent among these advances are X-ray crystallographic results identifying both oxidative and non-oxidative intermediates, rapid chemical quench followed by NMR detection of a several intermediates which are stable under acidic conditions, and circular dichroism detection of the 1',4'-imino tautomer of ThDP in some of the intermediates. Some of these methods also enable the investigator to determine the rate-limiting step in the complex series of steps.

Coenzymes↗
PubMed2006

Synthesis with good enantiomeric excess of both enantiomers of alpha-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases.

In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan, Biochemistry 40 (2001) 7369-7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21,473-21,482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 active-center variants. Both pyruvate and beta-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.

Acetone↗
PubMed2006

Theoretical model of interactions between ligand-binding sites in a dimeric protein and its application for the analysis of thiamine diphosphate binding to yeast transketolase.

The binding of thiamin diphosphate (ThDP) to yeast dimeric apotransketolase (apoTK) is accompanied by the appearance of a band in the absorption spectrum with maximum at 320 nm. The saturation function has been analyzed using a scheme that involves binding of ThDP to each subunit followed by the conformational transition of this subunit. It is assumed that the binding of ThDP to one subunit may affect the conformational transition of the other subunit. Rigorous mathematical expressions describing the dependence of the optical absorption on the total concentration of ThDP are first developed. Equilibrium constants and corresponding rate constants for the binding of ThDP to apoTK have been estimated. The negative cooperativity in the ThDP binding has been characterized by the function reflecting the dependence of the conformational change on the saturation of apoTK by ThDP.

Binding Sites↗
PubMed2004

The relationship between plasma and red cell concentrations of vitamins thiamine diphosphate, flavin adenine dinucleotide and pyridoxal 5-phosphate following elective knee arthroplasty.

BACKGROUND & AIMS: Water soluble vitamins B1, B2 and B6 are essential precursors for a wide variety of coenzymes involved in intermediary metabolism and their status is usually assessed from blood samples. The aim of the study was to examine the relationship between plasma and intra-cellular B-vitamins following the systemic inflammatory response of surgery. METHODS: Patients (n = 10) who underwent an elective knee arthroplasty, had venous blood samples withdrawn pre-operatively and at 12, 24, 48, 72 and 168 h after the start of surgery for the analysis of circulating concentrations of C-reactive protein and albumin and also plasma and/ or red cell thiamine diphosphate (TDP), flavin adenine dinucleotide (FAD), pyridoxal 5-phosphate (PLP) as indicators of vitamins B1, B2, and B6 status respectively. RESULTS: Pre-operative, baseline vitamin assessments were all within population reference ranges. Over the study period of 0-168 h there was a significant increase in circulating C-reactive protein concentrations (peak 48 h, P < 0.001) and a significant fall in albumin concentrations (trough 48 h, P < 0.001). Plasma FAD and PLP concentrations fell transiently (P < 0.001) by approximately 40% reaching their nadir at approximately 48 h. CONCLUSIONS: The results of the present study indicate that plasma concentrations of FAD and PLP are transiently reduced following an inflammatory insult and therefore unlikely to be a reliable measure of status in the presence of a systemic inflammatory response. It may be that during such a response red cell concentrations provide a more reliable measure.

Aged↗
PubMed2005

The relationship between plasma and red cell B-vitamin concentrations in critically-ill patients.

BACKGROUND AND AIMS: Low vitamin B-complex status has been associated with poorer outcome in critically-ill patients. However, these findings have been based on indirect methods. Using direct methods for assessing vitamin status, we examined the effect of B-complex vitamin supplementation by measuring plasma and red blood cell B1, B2 and B6-vitamin concentrations in critically-ill patients. METHODS: Thiamine diphosphate (TDP), flavin adenine dinucleotide (FAD) and pyridoxal phosphate (PLP) concentrations were measured in plasma and red cells of normal subjects (n=49) and ITU patients (n=41). RESULTS: Compared with the normal subjects, critically-ill patients had higher C-reactive protein and lower albumin concentrations (P<0.001). Also, plasma FAD and PLP were lower (P<0.001) and red cell concentrations of both were higher (P<0.01) in critically-ill patients. Critically-ill patients were grouped according to whether (n=23) or not (n=18) they had been supplemented with B-complex vitamins. Compared with non-supplemented group, the supplemented group had significantly higher red cell TDP and PLP concentrations (P<0.01). Plasma FAD and PLP concentrations did not differ significantly between the groups. CONCLUSIONS: The results of the present study suggest that direct measurements of red cell FAD and PLP are more responsive to supplementation than plasma measurements in the critically-ill patient.

Adult↗
PubMed2004

A new perspective on thiamine catalysis.

Our knowledge of thiamine-catalyzed ligase and lyase reactions has entered a new dimension. Significant achievements have been made in the field of enzymatic catalysis with the detection of hitherto unknown reaction types - extending the synthetic potential of known thiamine diphosphate (ThDP)-dependent enzymes - and the identification and characterization of new enzymes. As we learn more about ThDP-dependent enzymes, we find an ever-expanding range of reactions that they are able to catalyze and see increased amino acid sequence heterogeneity. By contrast, the three-dimensional structures of these enzymes, so far, seem to be highly similar. Non-enzymatic thiazolium and triazolium catalysts have also been developed, enhancing the scope of acyl anion chemistry.

Bacteria↗
PubMed2004

Kinetic study of the H103A mutant yeast transketolase.

Data from site-directed mutagenesis and X-ray crystallography show that His103 of holotransketolase (holoTK) does not come into contact with thiamin diphosphate (ThDP) but stabilizes the transketolase (TK) reaction intermediate, alpha,beta-dihydroxyethyl-thiamin diphosphate, by forming a hydrogen bond with the oxygen of its beta-hydroxyethyl group [Eur. J. Biochem. 233 (1995) 750; Proc. Natl. Acad. Sci. USA 99 (2002) 591]. We studied the influence of His103 mutation on ThDP-binding and enzymatic activity. It was found that mutation does not affect the affinity of the coenzyme to apotransketolase (apoTK) in the presence of Ca(2+) (a cation found in the native holoenzyme) but changes all the kinetic parameters of the ThDP-apoTK interaction in the presence of Mg(2+) (a cation commonly used in ThDP-dependent enzymes studies). It was concluded that the structures of TK active centers formed in the presence of Mg(2+) and Ca(2+) are not identical. Mutation of His103 led to a significant acceleration of the one-substrate reaction but a slow down of the two-substrate reaction so that the rates of both types of catalysis became equal. Our results provide evidence for the intermediate-stabilizing function of His103.

Alanine↗
PubMed2005

Determination of thiamine and its phosphate esters in rat tissues analyzed as thiochromes on a RP-amide C16 column.

A new reversed-phase chromatographic method is described for the separation and quantification of thiamine (T), thiamine monophosphate (TMP) and diphosphate (TDP) in rat tissues. Sample extraction with perchloric acid (HClO(4)) was found more suitable than extraction with trichloroacetic acid (TCA), as regards convenience and background fluorescence. Derivatization of thiamine vitamers to thiochromes was optimized and complete separation of TDP and TMP thiochromes was obtained on a RP-amide C16 column in isocratic elution, with T thiochrome eluting in less than 10 min. The precision and the accuracy of the HPLC procedure were assessed: ranging from 0.5 to 7.7% for intra-day and from 2.0 to 9.4% for inter-day precision, a recovery average of 101% was determined (range 90-111%). Mean values of recovery for TDP, TMP or T were 91, 96 and 90% for liver extracts, respectively. Analysis of vitamers in tissues of rat submitted to 8 days thiamin deficiency, followed by a 14 days repletion, showed a significant reduction of TPP after 8 days of depletion in liver (-67%), brains (-50%), kidneys (-60%), followed by a complete recovery upon repletion.

Animals↗
PubMed2005

The analysis of thiamin and its derivatives in whole blood samples under high pH conditions of the mobile phase.

In this study a protocol for the analysis of thiamin and thiamin coenzymes in whole blood was developed. Thiamin and its coenzymes are analyzed by reversed phase liquid chromatography (RPLC), precolumn derivatisation with alkaline potassium ferricyanide and fluorescence detection, all at pH 10. Under these relatively high pH conditions the detectability of the analytes and the robustness of the method were substantially improved. The use of a high pH resistant RPLC column was a crucial step in developing this analysis method. Reproducibility, linearity, recovery, detection limit and column robustness were investigated. The within-batch CV was <2.5%, the between-batch CV <4.5%. The method was linear far above the physiological relevant concentration level. Recovery was almost 100% on an average. The limit of quantification was 1 nmol/l. The robustness of the RPLC column proved to be very high. Up to 1500 injections hardly any substantial changes in retention times and efficiency were observed. In summary: Using a high pH resistant RPLC column resulted in a robust, sensitive and precise method for the analysis of total Vitamin B1 and especially of TDP.

Chromatography, Liquid↗
PubMed2006

Distinct modes of recognition of the lipoyl domain as substrate by the E1 and E3 components of the pyruvate dehydrogenase multienzyme complex.

Two-dimensional (15)N-heteronuclear single-quantum coherence (HSQC) NMR studies with a di-domain (lipoyl domain+ linker+ peripheral subunit-binding domain) of the dihydrolipoyl acetyltransferase (E2) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus allowed a molecular comparison of the need for lipoic acid to be covalently attached to the lipoyl domain in order to undergo reductive acetylation by the pyruvate decarboxylase (E1) component, in contrast with the ability of free lipoic acid to serve as substrate for the dihydrolipoyl dehydrogenase (E3) component. Tethering the lipoyl domain to the peripheral subunit-binding domain in a complex with E1 or E3 rendered the system more like the native enzyme complex, compared with the use of a free lipoyl domain, yet of a size still amenable to investigation by NMR spectroscopy. Recognition of the tethered lipoyl domain by E1 was found to be ensured by intensive interaction with the lipoyl-lysine-containing beta-turn and with residues in the protruding loop close to the beta-turn. The size and sequence of this loop varies significantly between species and dictates the lipoylated lipoyl domain as the true substrate for E1. In contrast, with E3 the main interaction sites on the tethered lipoyl domain were revealed as residues Asp41 and Ala43, which form a conserved sequence motif, DKA, around the lipoyl-lysine residue. No domain specificity is observed at this step and substrate channelling in the complex thus rests on the recognition of the lipoyl domain by the first enzyme, E1. The cofactor, thiamine diphosphate, and substrate, pyruvate, had distinct but contrasting effects on the E1/di-domain interaction, whereas NAD(+) and NADH had negligible effect on the E3/di-domain interaction. Tethering the lipoyl domain did not significantly change the nature of its interaction with E1 compared with a free lipoyl domain, indicative of the conformational freedom allowed by the linker in the movement of the lipoyl domain between active sites.

Amino Acid Motifs↗
PubMed2006

A versatile conformational switch regulates reactivity in human branched-chain alpha-ketoacid dehydrogenase.

The dehydrogenase/decarboxylase (E1b) component of the 4 MD human branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) is a thiamin diphosphate (ThDP)-dependent enzyme. We have determined the crystal structures of E1b with ThDP bound intermediates after decarboxylation of alpha-ketoacids. We show that a key tyrosine residue in the E1b active site functions as a conformational switch to reduce the reactivity of the ThDP cofactor through interactions with its thiazolium ring. The intermediates do not assume the often-postulated enamine state, but likely a carbanion state. The carbanion presumably facilitates the second E1b-catalyzed reaction, involving the transfer of an acyl moiety from the intermediate to a lipoic acid prosthetic group in the transacylase (E2b) component of the BCKDC. The tyrosine switch further remodels an E1b loop region to promote E1b binding to E2b. Our results illustrate the versatility of the tyrosine switch in coordinating the catalytic events in E1b by modulating the reactivity of reaction intermediates.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2005

How an enzyme answers multiple-choice questions.

Acetohydroxyacid synthase (AHAS) is the first common enzyme in the pathway for the biosynthesis of branched-chain amino acids. Interest in the enzyme has escalated over the past 20 years since it was discovered that AHAS is the target of the sulfonylurea and imidazolinone herbicides. However, several questions regarding the reaction mechanism have remained unanswered, particularly the way in which AHAS "chooses" its second substrate. A new method for the detection of reaction intermediates enables calculation of the microscopic rate constants required to explain this phenomenon.

Acetolactate Synthase↗
PubMed1987

Preliminary evaluation of cocarboxylase on myocardial protection of the rat heart.

The presence of cocarboxylase (CC) is essential for the oxidation of pyruvate to acetylcoenzyme A (acetyl-CoA) and its subsequent degradation by means of the Krebs cycle. We compared the effects of various concentrations of CC in a cardioplegic solution on the survival and hemodynamic and metabolic recovery of 23 isolated, working rat hearts subjected to 60 minutes of hypothermic (23 degrees C) ischemic arrest. Group 1 (N = 6) consisted of hearts infused with the basic cardioplegic solution (Tyers' solution with glucose), to which no CC was added. In group 2 (N = 6) CC was added at 0.1 ml/L to the cardioplegic solution. In group 3 (N = 5) CC was added at 1 ml/L, and in group 4 (N = 6) CC was added at 10 ml/L. The cardioplegic infusions were performed at a pressure of 40 mm Hg for 2 minutes just before arrest; 30 minutes later they were performed again for 1 minute. Only two hearts (33.3%) recovered in group 1 whereas five recovered in group 2, five (100%) in group 3, and five (83.3%) in group 4. The recovery of hemodynamic performance as a percentage of preischemic control values showed marked improvement in the CC groups, especially group 3, when compared with group 1. The metabolic variables in the CC groups were also markedly improved, with significantly (p less than .05) decreased levels of tissue lactate and increased levels of creatine phosphate compared with those in group 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗