The anomalous kinetics of coupled aspartate aminotransferase and malate dehydrogenase. Evidence for compartmentation of oxaloacetate.
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ABSTRACT:
SUBMITTER: Bryce CF
PROVIDER: S-EPMC1172624 | biostudies-other | 1976 Mar
REPOSITORIES: biostudies-other
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Project description:Kinetic measurements of the uptake of native mitochondrial aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro were carried out. The uptake of both the enzymes is essentially complete in 1 min and shows saturation characteristics. The rate of uptake of aspartate aminotransferase into mitochondria is decreased by malate dehydrogenase, and vice versa. The inhibition is exerted by isoenzyme remaining outside the mitochondria rather than by isoenzyme that has been imported. The thiol compound beta-mercaptoethanol decreases the rate of uptake of the tested enzymes; inhibition is a result of interaction of beta-mercaptoethanol with the mitochondria and not with the enzymes themselves. The rate of uptake of aspartate aminotransferase is inhibited non-competitively by malate dehydrogenase, but competitively by beta-mercaptoethanol. The rate of uptake of malate dehydrogenase is inhibited non-competitively by aspartate aminotransferase and by beta-mercaptoethanol. beta-Mercaptoethanol prevents the inhibition of the rate of uptake of malate dehydrogenase by aspartate aminotransferase. These results are interpreted in terms of a model system in which the two isoenzymes have separate but interacting binding sites within a receptor in the mitochondrial membrane system.
| S-EPMC1145008 | biostudies-other
Project description:Ca2+ signaling in mitochondria has been mainly attributed to Ca2+ entry to the matrix through the Ca2+ uniporter and activation of mitochondrial matrix dehydrogenases. However, mitochondria can also sense increases in cytosolic Ca2+ through a mechanism that involves the aspartate-glutamate carriers, extramitochondrial Ca2+ activation of the NADH malate-aspartate shuttle (MAS). Both pathways are linked through the shared substrate alpha-ketoglutarate (alphaKG). Here we have studied the interplay between the two pathways under conditions of Ca2+ activation. We show that alphaKG becomes limiting when Ca2+ enters in brain or heart mitochondria, but not liver mitochondria, resulting in a drop in alphaKG efflux through the oxoglutarate carrier and in a drop in MAS activity. Inhibition of alphaKG efflux and MAS activity by matrix Ca2+ in brain mitochondria was fully reversible upon Ca2+ efflux. Because of their differences in cytosolic calcium concentration requirements, the MAS and Ca2+ uniporter-mitochondrial dehydrogenase pathways are probably sequentially activated during a Ca2+ transient, and the inhibition of MAS at the center of the transient may provide an explanation for part of the increase in lactate observed in the stimulated brain in vivo.
| S-EPMC2652317 | biostudies-literature
Project description:Over the last few years, various inborn disorders have been reported in the malate aspartate shuttle (MAS). The MAS consists of four metabolic enzymes and two transporters, one of them having two isoforms that are expressed in different tissues. Together they form a biochemical pathway that shuttles electrons from the cytosol into mitochondria, as the inner mitochondrial membrane is impermeable to the electron carrier NADH. By shuttling NADH across the mitochondrial membrane in the form of a reduced metabolite (malate), the MAS plays an important role in mitochondrial respiration. In addition, the MAS maintains the cytosolic NAD+ /NADH redox balance, by using redox reactions for the transfer of electrons. This explains why the MAS is also important in sustaining cytosolic redox-dependent metabolic pathways, such as glycolysis and serine biosynthesis. The current review provides insights into the clinical and biochemical characteristics of MAS deficiencies. To date, five out of seven potential MAS deficiencies have been reported. Most of them present with a clinical phenotype of infantile epileptic encephalopathy. Although not specific, biochemical characteristics include high lactate, high glycerol 3-phosphate, a disturbed redox balance, TCA abnormalities, high ammonia, and low serine, which may be helpful in reaching a diagnosis in patients with an infantile epileptic encephalopathy. Current implications for treatment include a ketogenic diet, as well as serine and vitamin B6 supplementation.
| S-EPMC8362162 | biostudies-literature
Project description:Gene rv3722c of Mycobacterium tuberculosis is essential for in vitro growth, and encodes a putative pyridoxal phosphate-binding protein of unknown function. Here we use metabolomic, genetic and structural approaches to show that Rv3722c is the primary aspartate aminotransferase of M. tuberculosis, and mediates an essential but underrecognized role in metabolism: nitrogen distribution. Rv3722c deficiency leads to virulence attenuation in macrophages and mice. Our results identify aspartate biosynthesis and nitrogen distribution as potential species-selective drug targets in M. tuberculosis.
| S-EPMC7181641 | biostudies-literature
Project description:Intracellular malate-starch interconversion plays an important role in stomatal movements. We investigated whether malate or oxaloacetate from the cytosolic membrane side regulate anion channels in the plasma membrane of Arabidopsis thaliana guard cells. Physiological concentrations of cytosolic malate have been reported in the range of 0.4-3 mM in leaf cells. Guard cell patch clamp and two-electrode oocyte voltage-clamp experiments were pursued. We show that a concentration of 1 mM cytosolic malate greatly activates S-type anion channels in Arabidopsis thaliana guard cells. Interestingly, 1 mM cytosolic oxaloacetate also activates S-type anion channels. Malate activation was abrogated at 10 mM malate and in SLAC1 anion channel mutant alleles. Interestingly, malate activation of S-type anion currents was disrupted at below resting cytosolic-free calcium concentrations ([Ca2+ ]cyt ), suggesting a key role for basal [Ca2+ ]cyt signaling. Cytosolic malate was not able to directly activate or enhance SLAC1-mediated anion currents in Xenopus oocytes, whereas in positive controls, cytosolic NaHCO3 enhanced SLAC1 activity, suggesting that malate may not directly modulate SLAC1. Cytosolic malate activation of S-type anion currents was impaired in ost1 and in cpk5/6/11/23 quadruple mutant guard cells. Together these findings show that these cytosolic organic anions function in guard cell 'plasma membrane' ion channel regulation.
| S-EPMC6115288 | biostudies-literature
Project description:Because the mitochondrial inner membrane is impermeable to pyridine nucleotides, transport of reducing equivalents between the mitochondrial matrix and the cytoplasm relies on shuttle mechanisms, including the malate-aspartate shuttle and the glycerol-3-phosphate shuttle. These shuttles are needed for reducing equivalents generated by metabolic reactions in the cytosol to be oxidized via aerobic metabolism. Two isoenzymes of malate dehydrogenase (MDH) operate as components of the malate-aspartate shuttle, in which a reducing equivalent is transported via malate, which when oxidized to oxaloacetate, transfers an electron pair to reduce NAD to NADH. Several competing mechanisms have been proposed for the MDH-catalyzed reaction. This study aims to identify the pH-dependent kinetic mechanism for cytoplasmic MDH (cMDH) catalyzed oxidation/reduction of MAL/OAA. Experiments were conducted assaying the forward and reverse directions with products initially present, varying pH between 6.5 and 9.0. By fitting time-course data to various mechanisms, it is determined that an ordered bi-bi mechanism with coenzyme binding first followed by the binding of substrate is able to explain the kinetic data. The proposed mechanism is similar to, but not identical to, the mechanism recently determined for the mitochondrial isoform, mMDH. cMDH and mMDH mechanisms are also shown to both be reduced versions of a common, more complex mechanism that can explain the kinetic data for both isoforms. Comparing the simulated activity (ratio of initial velocity to the enzyme concentration) under physiological conditions, the mitochondrial MDH (mMDH) activity is predicted to be higher than cMDH activity under mitochondrial matrix conditions while the cMDH activity is higher than mMDH activity under cytoplasmic conditions, suggesting that the functions of the isoforms are kinetically tuned to their individual physiological roles.
| S-EPMC4302200 | biostudies-literature
Project description:1. A spectrophotometric assay of the rates of penetration of oxaloacetate and l-malate into mitochondria is described. The assay is based on the measurement of the oxidation of intramitochondrial NADH by oxaloacetate and of the reduction of intramitochondrial NAD(+) by malate. 2. The rate of entry of both oxaloacetate and l-malate into mitochondria is restricted, as shown by the fact that disruption of the mitochondrial structure can increase the rate of interaction between the dicarboxylic acids and intramitochondrial NAD(+) and NADH by between 100- and 1000-fold. 3. The rates of entry of oxaloacetate and malate into liver, kidney and heart mitochondria increased by up to 50-fold on addition of a source of energy, either ascorbate plus NNN'N'-tetramethyl-p-phenylenediamine aerobically, or ATP anaerobically. 4. In the absence of a source of energy the changes in the concentrations of intramitochondrial NAD(+) and NADH brought about by the addition of l-malate or oxaloacetate were followed by parallel changes in the concentrations of NADP(+) and NADPH, indicating the presence in the mitochondria of an energy-independent transhydrogenase system. 5. The results are discussed in relation to the hypothesis that malate acts as a carrier of reducing equivalents between mitochondria and cytoplasm.
| S-EPMC1198718 | biostudies-other
Project description:Aspartate aminotransferases have been cloned and expressed from Crithidia fasciculata, Trypanosoma brucei brucei, Giardia intestinalis, and Plasmodium falciparum and have been found to play a role in the final step of methionine regeneration from methylthioadenosine. All five enzymes contain sequence motifs consistent with membership in the Ia subfamily of aminotransferases; the crithidial and giardial enzymes and one trypanosomal enzyme were identified as cytoplasmic aspartate aminotransferases, and the second trypanosomal enzyme was identified as a mitochondrial aspartate aminotransferase. The plasmodial enzyme contained unique sequence substitutions and appears to be highly divergent from the existing members of the Ia subfamily. In addition, the P. falciparum enzyme is the first aminotransferase found to lack the invariant residue G197 (P. K. Mehta, T. I. Hale, and P. Christen, Eur. J. Biochem. 214:549-561, 1993), a feature shared by sequences discovered in P. vivax and P. berghei. All five enzymes were able to catalyze aspartate-ketoglutarate, tyrosine-ketoglutarate, and amino acid-ketomethiobutyrate aminotransfer reactions. In the latter, glutamate, phenylalanine, tyrosine, tryptophan, and histidine were all found to be effective amino donors. The crithidial and trypanosomal cytosolic aminotransferases were also able to catalyze alanine-ketoglutarate and glutamine-ketoglutarate aminotransfer reactions and, in common with the giardial aminotransferase, were able to catalyze the leucine-ketomethiobutyrate aminotransfer reaction. In all cases, the kinetic constants were broadly similar, with the exception of that of the plasmodial enzyme, which catalyzed the transamination of ketomethiobutyrate significantly more slowly than aspartate-ketoglutarate aminotransfer. This result obtained with the recombinant P. falciparum aminotransferase parallels the results seen for total ketomethiobutyrate transamination in malarial homogenates; activity in the latter was much lower than that in homogenates from other organisms. Total ketomethiobutyrate transamination in Trichomonas vaginalis and G. intestinalis homogenates was extensive and involved lysine-ketomethiobutyrate enzyme activity in addition to the aspartate aminotransferase activity. The methionine production in these two species could be inhibited by the amino-oxy compounds canaline and carboxymethoxylamine. Canaline was also found to be an uncompetitive inhibitor of the plasmodial aspartate aminotransferase, with a K(i) of 27 microm.
| S-EPMC95336 | biostudies-literature
Project description:Diffluoro-oxaloacetate behaves as a competitive inhibitor of 2-oxoglutarate and as an uncompetitive inhibitor with respect to aspartate in steady-state kinetic experiments with cytoplasmic aspartate transaminase. In the presence of high concentrations of aspartate transaminase, difluoro-oxaloacetate is slowly transaminated to difluoro-aspartate, suggesting its use as a kinetic probe to study the reactions of the aminic form of the enzyme.
| S-EPMC1164515 | biostudies-other
Project description:Aspartate: 2-oxoglutarate aminotransferase from the anaerobic protozoon Trichomonas vaginalis was purified to homogeneity and characterized. It is a dimeric protein of overall Mr approx. 100000. Only a single isoenzyme was found in T. vaginalis. The overall molecular and catalytic properties have features in common with both the vertebrate cytoplasmic and mitochondrial isoenzymes. The purified aspartate aminotransferase from T. vaginalis showed very high rates of activity with aromatic amino acids as donors and 2-oxoglutarate as acceptor. This broad-spectrum activity was restricted to aromatic amino acids and aromatic 2-oxo acids, and no significant activity was seen with other common amino acids, other than with the substrates and products of the aspartate: 2-oxoglutarate aminotransferase reaction. Co-purification and co-inhibition, by the irreversible inhibitor gostatin, of the aromatic amino acid aminotransferase and aspartate aminotransferase activities, in conjunction with competitive substrate experiments, strongly suggest that a single enzyme is responsible for both activities. Such high rates of aromatic amino acid aminotransferase activity have not been reported before in eukaryotic aspartate aminotransferase.
| S-EPMC1152940 | biostudies-other