Peter F. Nixon

Effect of substrate decomposition on the spectrophotometric assay of dihydrofolate reductase

Abstract 1. (1) Both dihydrofolate and NADPH were found to decompose at a significant rate at pH values below 7, but decomposition of the latter compound was considerably faster than dihydrofolate decomposition and at pH 4.0 it was so rapid that it precluded reliable assay of dihydrofolate reductase. 2. (2) NADPH decomposition at low pH could be decreased by lowering the buffer concentration and by substituting acetate for citrate buffer. Dihydrofolate decomposition was retarded by thiols or ascorbate and increased by EDTA. 3. (3) At pH 4.5 the decomposition of either substrate at the normal assay concentration in the presence of 5 m M mercaptoethanol and 50 m M acetate buffer was insufficient in 10 min at 37° to affect the subsequent assay of dihydrofolate reductase. 4. (4) Thiols decreased the absorbancy change at 340 mμ which accompanies dihydrofolate decomposition by reacting with a pteridine decomposition product. The properties of the latter suggest that it is 2-amino-7,8-dihydro-4-hydroxypteridine-6-carboxaldehyde. 5. (5) The solubility of microcrystalline dihydrofolate at low pH has been determined at 25° and 37°. 6. (6) Redetermination of the molar absorbancy change in the dihydrofolate reductase reaction has yielded a value of 12,300 ± 320.

Properties and functions of the thiamin diphosphate dependent enzyme transketolase

This review highlights recent research on the properties and functions of the enzyme transketolase, which requires thiamin diphosphate and a divalent metal ion for its activity. The transketolase-catalysed reaction is part of the pentose phosphate pathway, where transketolase appears to control the non-oxidative branch of this pathway, although the overall flux of labelled substrates remains controversial. Yeast transketolase is one of several thiamin diphosphate dependent enzymes whose three-dimensional structures have been determined. Together with mutational analysis these structural data have led to detailed understanding of thiamin diphosphate catalysed reactions. In the homodimer transketolase the two catalytic sites, where dihydroxyethyl groups are transferred from ketose donors to aldose acceptors, are formed at the interface between the two subunits, where the thiazole and pyrimidine rings of thiamin diphosphate are bound. Transketolase is ubiquitous and more than 30 full-length sequences are known. The encoded protein sequences contain two motifs of high homology; one common to all thiamin diphosphate-dependent enzymes and the other a unique transketolase motif. All characterised transketolases have similar kinetic and physical properties, but the mammalian enzymes are more selective in substrate utilisation than the nonmammalian representatives. Since products of the transketolase-catalysed reaction serve as precursors for a number of synthetic compounds this enzyme has been exploited for industrial applications. Putative mutant forms of transketolase, once believed to predispose to disease, have not stood up to scrutiny. However, a modification of transketolase is a marker for Alzheimers disease, and transketolase activity in erythrocytes is a measure of thiamin nutrition. The cornea contains a particularly high transketolase concentration, consistent with the proposal that pentose phosphate pathway activity has a role in the removal of light-generated radicals.

Effective absorption and utilization of oral formyltetrahydrofolate in man

Abstract Reversal of the effects of antifolates requires effective expansion of the body pool of reduced folate coenzymes. In the past, this has required parenteral use of 5-formyltetrahydrofolate. By use of Radio-labeled 5-formyltetrahydrofolate, we showed that its oral administration also expands the body reduced folate pool. After oral administration of 5-formyl-14C-tetrahydrofolate-3H to fasting subjects, the labels appeared in the serum, peaking at 60 minutes. Chromatographic analysis showed that the labeled serum folate was principally the naturally occurring 5-methyltetrahydrofolate-3H. Close to 90 per cent of orally administered 5-formyltetrahydrofolate appeared to be absorbed. The most constant labeled urinary folate was found to be 10-formyltetrahydrofolate or 5, 10-methenyltetrahydrofolate. Renal excretion of labeled 5-formyltetrahydrofolate occurred at undetectable serum levels whereas renal excretion of labeled 5-methyltetrahydrofolate was proportional to its serum concentration.

An erythrocyte transketolase isoenzyme pattern associated with the Wernicke-Korsakoff syndrome.

Abstract. Two techniques were used to seek variants of human erythrocyte transketolase and to test for any association of the Wernicke‐Korsakoff syndrome, a thiamin‐deficiency disease, with a particular variant of this thiamin‐dependent enzyme. Apparent Km values for the cofactor thiamin diphosphate were similar for patients and controls. However, isoelectric focussing separated erythrocyte transketolase into different isoenzymes characterized by pI values in the range 6·6–9·2. Six distinct patterns of isoenzymes were found in thirty‐six healthy control subjects. The isoenzyme pattern for thirty‐nine out of forty‐two patients suffering from the Wernicke‐Korsakoff syndrome was identical to a pattern found in only eight of thirty‐six control subjects, a highly significant association (P < 0·001). This association suggests that a variant transketolase and thiamin deficiency together contribute to the pathogenesis of the brain damage of the Wernicke‐Korsakoff syndrome by some mechanism independent of apparent Km values for thiamin diphosphate.

Molecular Evolutionary Analysis of the Thiamine-Diphosphate-Dependent Enzyme, Transketolase

Abstract. Members of the transketolase group of thiamine-diphosphate-dependent enzymes from 17 different organisms including mammals, yeast, bacteria, and plants have been used for phylogenetic reconstruction. Alignment of the amino acid and DNA sequences for 21 transketolase enzymes and one putative transketolase reveals a number of highly conserved regions and invariant residues that are of predicted importance for enzyme activity, based on the crystal structure of yeast transketolase. One particular sequence of 36 residues has some similarities to the nucleotide-binding motif and we designate it as the transketolase motif. We report further evidence that the recP protein from Streptococcus pneumoniae might be a transketolase and we list a number of invariant residues which might be involved in substrate binding. Phylogenies derived from the nucleotide and the amino acid sequences by various methods show a conventional clustering for mammalian, plant, and gram-negative bacterial transketolases. The branching order of the gram-positive bacteria could not be inferred reliably. The formaldehyde transketolase (sometimes known as dihydroxyacetone synthase) of the yeast Hansenula polymorpha appears to be orthologous to the mammalian enzymes but paralogous to the other yeast transketolases. The occurrence of more than one transketolase gene in some organisms is consistent with several gene duplications. The high degree of similarity in functionally important residues and the fact that the same kinetic mechanism is applicable to all characterized transketolase enzymes is consistent with the proposition that they are all derived from one common ancestral gene. Transketolase appears to be an ancient enzyme that has evolved slowly and might serve as a model for a molecular clock, at least within the mammalian clade.

Measurement of Michaelis constant for human erythrocyte transketolase and thiamin diphosphate.

Human erythrocyte transketolase could be resolved from thiamin diphosphate (TDP) by acidification of the ammonium sulfate precipitate to pH 3.5, but not by other tested procedures. Resolution was 98% by chemical measurement of residual thiamin and 95% by residual enzyme activity. Reconstitution of the resolved preparation by incubation with TDP was dependent upon TDP concentration, duration, temperature, and the presence of dithiothreitol. At low TDP concentrations, 1 h was required for maximum activation; kinetic analysis then yielded an apparent Km value for TDP of 65 nM (SD 14 nM) from 100 erythrocyte lysates and similar values for reconstituted resolved preparations previously purified 400-fold and 10,000-fold. Velocity data obtained by transketolase assays in which the TDP was added to resolved preparations simultaneously with substrates yielded an apparent Km value for TDP of 2.3 microM (SD 1.6 microM) from 114 erythrocyte lysates and similar values for purified preparations. The recovery of activity following resolution and reconstitution ranged from 21 to 60% from lysates and 38 to 70% from purified preparations. Residual ammonium sulfate up to 4.9 mM decreased the apparent Km value for TDP, while a concentration of 11.3 mM increased the value in a manner competitive with TDP and with an apparent Ki value of 2.3 mM. The spectrophotometric assay of transketolase activity was greatly affected by storage of frozen solutions of the substrate ribose 5-phosphate.

Progression of neurological disease in thiamin-deficient rats is enhanced by ethanol

The clinical and neuropathological consequences of either ethanol consumption or thiamin deficiency or both were examined in Wistar rats aged nine weeks divided into five groups and fed one of the following diets: a thiamin-replete (control) diet (A): a thiamin-fortified diet with water (B) or 15% ethanol (C); or a thiamin-deficient diet with water (D) or 15% ethanol (E). Rats fed diets A, B or C for 35 weeks showed no clinical signs of neurological disease at any stage and no significant brain pathology when harvested. Rats fed diets D and E progressed through a common sequence of clinical signs of neurological disease typical of acute thiamin deficiency, viz loss of coat condition, ataxia, opisthotonus and ultimately death within 10-23 weeks. The onset and progression of these stages of neurological disease were significantly earlier and faster (p less than 0.001 for proportion of opisthotonic and ataxic animals at weeks 10 and 15) in the thiamin-deficient rats that received ethanol than in those that did not. At death, the brain pathology in these two groups was limited and similar.

Effect of chronic alcohol ingestion on hepatic folate distribution in the rat

The mechanism by which ethanol impairs folate metabolism remains uncertain. In the present study, we used our new technique (affinity/HPLC) for folate analysis to study the effect of chronic alcohol ingestion on the content and distribution of folates in livers. Twelve male Sprague-Dawley rats (180 g) were divided into two groups, and fed for 4 weeks with Lieber-DeCarli semi-liquid isocaloric diets, with and without 5% ethanol. Livers were extracted in boiling, pH 9.3 borate buffers containing ascorbate/dithioerythritol. Folates in the supernatant fractions were purified by affinity chromatography and analyzed using ion pair high performance liquid chromatography. The data obtained showed that hepatic folate distribution in alcohol-treated rats differed from that of control animals in two ways. Livers from the ethanol-fed rats, when compared with those from control rats, exhibited increases in the percent concentrations of methylated tetrahydrofolates (21.46 +/- 2.21 vs 14.8 +/- 1.23), decreases in the percent concentrations of formylated tetrahydrofolates (25.62 +/- 4.02 vs 46.18 +/- 2.65) and higher concentrations of unsubstituted tetrahydrofolates (52.91 +/- 3.84 vs 38.88 +/- 2.50). In addition, alcohol ingestion was associated with longer glutamate chains of the folate molecules, characterized by lower relative concentrations of pentaglutamyl folates (29 vs 48%), and higher relative concentrations of hexa- and heptaglutamyl folates (55 vs 46% and 15 vs 6%) when compared with controls. The data are discussed in relation to the possibility that alcohol exerts its effect through: (1) inhibition of B12-dependent methyl transfer from methyltetrahydrofolate to homocysteine; (2) diversion of formylated tetrahydrofolates toward serine synthesis; and (3) interaction of acetaldehyde with tetrahydrofolates, thereby interfering with folate coenzyme metabolism.

Aspartate-27 and glutamate-473 are involved in catalysis by Zymomonas mobilis pyruvate decarboxylase

Zymomonas mobilis pyruvate decarboxylase (EC 4.1.1.1) was subjected to site-directed mutagenesis at two acidic residues near the thiamin diphosphate cofactor in the active site. Asp-27 was changed to Glu or Asn, and Glu-473 was mutated to Asp (E473D) or Gln (E473Q). Each mutant protein was purified to near-homogeneity, and the kinetic and cofactor-binding properties were compared with those of the wild-type protein. Despite the very conservative nature of these alterations, all mutants had a very low, but measurable, specific activity ranging from 0.025% (E473Q) to 0.173% (E473D) of the wild type. With the exception of E473Q, the mutants showed small decreases in the affinity for thiamin diphosphate, and binding of the second cofactor (Mg2+) was also weakened somewhat. With E473Q, both cofactors seemed to be very tightly bound so that they were not removed by the treatment that was effective for the wild-type enzyme and other mutant forms. All mutants showed minor changes in the Km for substrate, but these alterations did not account for the low activities. These low specific activities, accompanied by little change in the Km for pyruvate, are consistent with a quantitative model of the catalytic cycle in which the main effect of the mutations is to slow the decarboxylation step with a minor change in the rate constant for pyruvate binding.

Glucose loading precipitates acute encephalopathy in thiamin-deficient rats.

A rat model of glucose-precipitated Wernickes encephalopathy (WE) has been developed in which glucose loading (10 g/kg, i.p.) of ataxic thiamin-deficient (TD) rats induced episodes of gross neurological dysfunction and sometimes death. The acute effects of a glucose load on the neurological state of thiamin-replete control and TD rats were assessed by scoring of clinical observations and performance measured on a moving belt (MB) apparatus at 30 min intervals for 2 hr after the challenge. Glucose loading or saline treatment (2.5 mL, i.p.) had no significant behavioural or clinical consequences when administered to controls or rats fed TD diet for <21 days. Glucose loading of ataxic rats fed TD diet for 28-35 days precipitated episodes of gross ataxia and signs of advanced neurological dysfunction (e.g. loss of righting reflex and hyperexcitability) leading to significant increases in the Ataxia (p<0.05) and Advanced Sign (p<0.05) scores within 2 hr after the challenge. Simultaneously, the performance of these animals on the MB decreased 10-fold. Regular glucose challenges significantly increased the rate of progression of disease in TD rats when compared with untreated TD rats. This model may be useful for the further investigation of the pathogenesis of WE at the molecular level.