We hypothesized that the exercise-induced increase in muscle tricarboxylic acid (TCA) cycle intermediates (TCAI) would be lower after aerobic training (TR), due to a better match between pyruvate production and subsequent oxidation and lower flux through the alanine aminotransferase (AAT) reaction. Eight men [22 ± 1 y; maximal aerobic capacity (V02max) = 3.9 ± 0.2 L/min] cycled at 75% of their pre-TR V02max to exhaustion (Exh), before and after 7 wk ofTR (1 hr/d, 5 d/wk). Muscle biopsies (v. lateralis) were obtained at rest, 5 min of exercise and Exh. The effect ofTR was evidenced by an increased time to fatigue (91 ± 6 vs 42 ± 6 min), increases in resting [glycogen] and citrate synthase maximal activity, and decreases in glycogen degradation, lactate accumulation and phosphocreatine utilization during exercise. The sum of 4 measured TCAI was similar between trials at rest, but lower after 5 min of exercise post- TR (2. 7 ± 0.2 vs 4.3 ± 0.2 mmol.kg-1 dw, P<0.05). Importantly, the [TCAI] at Exh post- TR (2.9 ± 0.2 mmol.kg-1 dw) was not different compared to 5 min of exercise and thus fatigue was not attributable to a decline in TCAI. The net change in glutamate (Post: 4.5 ± 0.7 vs Pre: 7.7 ± 0.6 mmol.kg-1 dw) and alanine (Post: 3.3 ± 0.2 vs Pre: 5.6 ± 0.3 mmol.kg-1 dw) from Rest-5 min of exercise was attenuated post-TR (P<0.05), which is consistent with lower flux through the AA T reaction. We conclude that changes in muscle TCAI during exercise are not causally related to aerobic energy provision. / Thesis / Master of Science (MSc)

Extreme thermophiles and hyperthermophiles are microorganisms capable of growing optimally at 65-79??C and 80??C plus, respectively. Many of the enzymes isolated from them are thermostable, which makes them a potential resource for research and industrial applications. An increasing number of hyper/thermophiles is shown to be able to produce ethanol as an end-metabolite. Despite characterization of many alcohol dehydrogenases (ADHs) with a potential role in the production of ethanol, to date there has been no significant progress in identifying the enzymes responsible for the production of acetaldehyde, which is an intermediate in production of ethanol from pyruvate.<br> Pyruvate decarboxylase (PDC encoded by pdc) is a thiamine pyrophosphate (TPP)-containing enzyme responsible for conversion of pyruvate to acetaldehyde in many mesophilic organisms. However, no pdc/PDC homolog has yet been found in fully sequenced genomes of hyper/thermophiles. The only PDC activity reported in hyperthermophiles is a bifunctional, TPP- and CoA-dependent pyruvate ferredoxin oxidoreductase (POR)/PDC enzyme from the hyperthermophilic archaeon Pyrococcus furiosus.<br> The bifunctional and TPP-containing POR/PDC enzyme was isolated and characterized from the ethanol-producing hyperthermophilic archaeon Thermococcus guaymasensis (Topt=88??C), as well as the bacteria Thermotoga hypogea (Topt=70??C) and Thermotoga maritima (Topt=80??C). The T. guaymasensis enzyme was purified anaerobically to homogeneity as judged by SDS-PAGE analysis. POR and PDC activities were co-eluted from each of the chromatographic columns, and the ratio of POR to PDC activities remained constant throughout the purification steps. All of the enzyme activities were CoA- and TPP-dependent and highly sensitive toward exposure to air. The apparent kinetic parameters were determined for the main substrates, including pyruvate and CoA for each activity. Since the genome sequence of T. guaymasensis and T. hypogea were not available, sequences of the genes encoding POR were determined via primer walking and inverse PCR.<br> A novel enzyme capable of catalyzing the production of acetaldehyde from pyruvate in hyperthermophiles was also characterized. The enzyme contained TPP and flavin and was expressed as recombinant histidine-tagged protein in the mesophilic host Escherichia coli. The new enzyme was a bifunctional enzyme catalyzing another reaction as the major reaction besides catalyzing the non-oxidative decarboxylation of pyruvate to acetaldehyde.<br> Another enzyme known to be involved in catalysis of acetaldehyde production from pyruvate is CoA-acetylating acetaldehyde dehydrogenase (AcDH encoded by mhpF and adhE). Pyruvate is oxidized into acetyl-CoA by either POR or pyruvate formate lyase (PFL), and AcDH catalyzes the reduction of acetyl-CoA to acetaldehyde. AcDH is present in some mesophilic (such as clostridia) and thermophilic bacteria (e.g. Geobacillus and Thermoanaerobacter). However, no AcDH gene or protein homologs could be found in the released genomes of hyperthermophiles. Moreover, no such activity was detectable from the cell-free extracts of different hyperthermophiles used in this study.<br> In conclusion, no commonly-known PDCs was found in hyperthermophiles, but two types of acetaldehyde-producing enzymes were present in various bacterial and archaeal hyperthermophiles. Although the deduced amino acid sequences from different hyperthermophiles are quite similar, the levels of POR and PDC activities appeared to vary significantly between the archaeal and bacterial enzymes, which most likely reflects the different physiological implications of each activity.

No description available.

In the anaerobic protozoon <i>Trichomonas vaginalis</i>, the oxidative decarboxylation of pyruvate is catalysed in a CoA-dependent reaction by pyruvate: ferredoxin oxidoreductase (PFOR). This enzyme has been identified as a potential target for the development of a relatively non-toxic anti-trichomonal agent. 1. <i>T. vaginalis</i> PFOR was localised in the hydrogenosomal membrane fraction, and could be solubilised by buffer of high ionic strength. A high salt concentration was required to prevent aggregation of PFOR. These results suggested that PFOR was either an extrinsic protein bound to the hydrogenosomal membrane or that the enzyme exists <i>in vivo</i> in the hydrogenosomal matrix in an aggregated state. PFOR was solubilised and purified to homogeneity, the most _ffective step being salting-out chromatography on Sepharose 4B. Low recoveries of active enzyme were caused by inactivation by oxygen and the irreversible loss of thiamin pyrophosphate (TPP). 2. PFOR is a dimeric enzyme of overall M<SUB>r</SUB>240000. The enzyme contains 0.5 mol of TPP per mol of dimer, and equivalent amounts of non-haem iron and acid-labile sulphur, consistent with the presence of two [4Fe-4S] centres per enzyme molecule. Flavin nucleotides and lipoic acid are absent. PFOR from <i>T. vaginalis</i> is therefore broadly similar to the 2-oxo acid:ferredoxin (flavodoxin) oxidoreductases purified from bacterial sources, and clearly different from the 2-oxo acid dehydrogenase multienzyme complexes which occur in aerobic organisms. 3. A steady-state kinetic analysis of purified PFOR demonstrated that the enzyme obeyed Bi Bi Ping Pong kinetics except at very high CoA concentrations, where substrate inhibition occurred. The inhibition produced by the product of acetyl-CoA, in the presence of saturating CoA, was competitive with respect to pyruvate. In the absence of CoA, stoichiometric amounts of pyruvate were decarboxylated by PFOR. These results suggest that decarboxylation, formation of the stable imtermediate and its reaction with CoA to form acetyl-CoA all take place at one active site. 4. Spectroscopic investigations using electron paramagnetic resonance indicated that the stable intermediate formed after pyruvate decarboxylation was a free-radical species. This substrate-based radical is proposed to arise by the transfer of a single electron from the initial decarboxylation product to a [4Fe-4S] centre. The free-radical signal was greatly diminished if the enzyme was subsequently incubated with CoA, suggesting that it represents a real catalytic intermediate. 5. <i>T. vaginalis</i> PFOR was inactivated by incubation with pyruvate alone, a reaction the enzyme has in common with the <i>E. coli</i> pyruvate dehydrogenase (PDH) complex and yeast pyruvate decarboxylase, suggesting similarities between these enzymes at least in the initial formation of the decarboxylated intermediate, presumed to be the enamine of hydroxyethyl-TPP. The conjugated 2-oxo acid, (E)-4-(-chorophenyl)-2-oxo-3-butenoic acid, was an irreversible inhibitor of <i>T. vaginalis</i> PFOR and yeast pyruvate decarboxylase, a result taken to reflect the initial formation of an enamine intermediate in each case. 6. 3-hydroxypyruvate was a potent irreversible inhibitor of <i>T. vaginalis</i> PFOR. The observation that 3-hydroxypyruvate was also an alternative substrate for pyruvate in the overall reaction suggested that it might be acting as a mechanism-based inactivator. 3-hydroxypyruvate was ineffective against <i>E. coli</i> PDH complex suggesting an interesting difference in active site geometry that might be exploited for potential drug design.

No description available.

No description available.

ABSTRACT Pyruvate kinase is an essential enzyme in the anaerobic glycolytic pathway of the erythrocyte. The clinical presentation of this enzyme deficiency is due to the haemolytic process that results from the inability of erythrocytes to generate sufficient ATP. Although pyruvate kinase and glucose-6-phosphate dehydrogenase deficiencies comprise more than 90% of all reported red cell enzyme disorders worldwide, the epidemiology of the disease in South Africa is unknown and there is no assay for pyruvate kinase activity currently being used in South Africa. This report describes the implementation of screening and quantitative assays for pyruvate kinase activity in the Red Cell Membrane Unit at the University of the Witwatersrand Medical School / NHLS. The accuracy, precision and reproducibility of the assay were verified. Furthermore, a patient with pyruvate kinase deficiency was confirmed and found to have 15% of normal enzyme activity at 37oC. The genetic abnormality was identified as a homozygous G1529A point mutation in exon 11 of the pyruvate kinase gene and to the candidate’s knowledge is the first mutation described in a South African kindred. The patient’s mother was heterozygous for the G1529A mutation and demonstrated an enzyme activity of 58% of normal at 37oC.

"June, 2003" / Includes bibliographical references (leaves 169-200) / viii, 215 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Science, Discipline of Biochemistry, 2004

This thesis examines Escherichia coli pyruvate kinase type 1 (PK1), a regulatory enzyme core to energy metabolism. Specifically, this thesis characterises a series of mutations in PK1 that were found when populations of E. coli were evolved in a glucose-limited environment for 20,000 generations. The gene pykF, which codes for the PK1 enzyme, was found to have developed nonsynonymous mutations in all replicate populations. Although the mutations at the nucleotide level were not the same (i.e. not parallel), it is not clear whether parallel adaptation exists at the protein structure/function level. This study aimed to address this question by investigating the kinetic and biophysical properties of the wild-type and seven mutant enzymes. The recombinant wild-type PK1 enzyme used in this study was found to have steady state kinetics consistent with those previously reported. Unlike the rabbit kidney PK enzyme, E. coli PK1 was shown to have a very tight tetrameric structure (picomolar range), which was not affected by the enzyme’s substrates (PEP and ADP), or the allosteric effector (FBP), as judged by analytical ultracentrifugation with fluorescence detection. The mutated residues were highly conserved, and found to fall loosely into three groups: those at the active site (P70T, P70Q and D127N); those at the subunit interface (I264F, A301T and A301S); and at the allosteric binding site (G381A). The seven mutated PK1 enzymes were obtained by mutagenesis followed by protein purification. Steady state kinetic analysis showed that the mutated enzymes displayed a variety of functional changes, suggesting that the populations had not evolved in a parallel manner at the enzyme structure/function level. Mutations within the active site (P70T, P70Q and D127N) all showed a decrease in catalytic potency. P70 is located at the hinge connecting the A and B domains, which forms the active site. PK1-P70Q showed strong cooperative binding to PEP, similar to the wild-type enzyme, in the absence of FBP, whereas PK1-P70T had little cooperativity, suggesting changes in the active site. PK1-D127N showed severely attenuated activity, suggesting, for the first time, that this residue is essential for catalysis. Mutations at the subunit interface (I264F, A301T and A301S) all showed altered allosteric regulation, suggesting that this interface is important in the FBP allosteric response. PK1-I264F, which had lower activity, but greater affinity for PEP, displayed a decreased α-helix content (as judged by CD), indicating that a subunit interface helix that includes this residue had altered. Despite still having a similar response to FBP, PK1-G381A showed an increased affinity for PEP, which, together with an increased α-helix content, suggests that this mutation had changed the structure of the FBP binding domain. None of the mutated enzymes showed altered quaternary structure. Although the populations evolved parallel changes with respect to cell physiology, fitness, and gene expression, this study suggests, for the first time, that the populations have not evolved in a parallel way with respect to protein structure and function.

Bibliography: leaves 153-198. / viii, 198, [117] leaves, [14] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1999?