Mutation of the fumarase gene in two siblings with progressive encephalopathy and fumarase deficiency.

We report an inborn error of the tricarboxylic acid cycle, fumarase deficiency, in two siblings born to first cousin parents. They presented with progressive encephalopathy, dystonia, leucopenia, and neutropenia. Elevation of lactate in the cerebrospinal fluid and high fumarate excretion in the urine led us to investigate the activities of the respiratory chain and of the Krebs cycle, and to finally identify fumarase deficiency in these two children. The deficiency was profound and present in all tissues investigated, affecting the cytosolic and the mitochondrial fumarase isoenzymes to the same degree. Analysis of fumarase cDNA demonstrated that both patients were homozygous for a missense mutation, a G-955-->C transversion, predicting a Glu-319-->Gln substitution. This substitution occurred in a highly conserved region of the fumarase cDNA. Both parents exhibited half the expected fumarase activity in their lymphocytes and were found to be heterozygous for this substitution. The present study is to our knowledge the first molecular characterization of tricarboxylic acid deficiency, a rare inherited inborn error of metabolism in childhood.

A comparative study of the thermal inactivation of cytosolic and mitochondrial aspartate aminotransferases.

Rates of irreversible thermal inactivation of cytosolic and mitochondrial aspartate aminotransferases were measured over a large temperature range. Inactivation occurred by different kinetic pathways at high and low temperature with a transition point at about 60 degrees C. This suggests that the isoenzymes exist in different conformations above and below that temperature. Discontinuities in plots of ln(Vmax) against 1/T provided confirmatory evidence for this hypothesis. Activation parameters (deltaH and deltaS) for the thermal inactivation process were calculated in the high and low temperature ranges. At high temperature the greater rate of inactivation of the mitochondrial isoenzyme is determined largely by a high value of deltaS. This more than compensates for the fact that the deltaH is also greater for the mitochondrial isoenzyme indicative of greater intramolecular stabilising interactions compared with the cytosolic form. Thus the relative rates of inactivation are determined by the nature of the transition states rather than by intramolecular interactions in the folded proteins. At lower temperatures the kinetic stabilities of the isoenzymes reverse with the mitochondrial isoenzyme inactivating more slowly. This is largely because of a considerably smaller deltaS at low temperature which no longer compensates for the greater deltaH compared with the cytosolic isoenzyme.

Purification and structural comparisons of the cytosolic and mitochondrial isoenzymes of fumarase from pig liver.

A method has been developed for the purification of cytosolic and mitochondrial isoenzymes of fumarase from total homogenates of pig liver. Separation of the isoenzymes from one another was achieved using chromatofocusing. The isoenzymes were pure as judged by production of single bands on electrophoresis in the presence of sodium dodecyl sulphate; they appeared to have identical or very similar subunit molecular weights. The isoenzymes differed in electrophoretic properties under nondenaturing conditions. One-dimensional peptide maps of fragments produced from the two isoenzymes by chemical cleavage at cysteine residues were identical; maps obtained after digestion with the V8 proteinase from Staphylococcus aureus showed small differences at short times of digestion which could have reflected variations in rates of hydrolysis rather than structural differences. However, two-dimensional peptide maps of digests obtained by treatment of the isoenzymes with trypsin followed by chymotrypsin had 58 peptides in common, but showed two peptides unique to the mitochondrial isoenzyme and five peptides unique to the cytosolic form. Using the dansylation procedure, the mitochondrial isoenzyme was shown to have N-terminal alanine and the cytosolic form to have N-terminal glutamic acid or glutamine. We conclude that the isoenzymes of fumarase are identical over nearly all of their amino acid sequences but differ at their N-termini; the extent of these differences is yet to be established. These results are consistent with the claim (Edwards, Y.H. and Hopkinson,D.A. (1979) Ann. Human Genet. Lond. 42, 303-313) that the isoenzymes are determined at the same genetic locus, but they raise interesting questions about the biosynthesis of the isoenzymes.

Partial amino-acid sequence and cysteine reactivities of cytosolic aspartate aminotransferase from horse heart.

Cytosolic aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) from horse heart has five cysteine residues, two of which can be titrated with 5,5'-dithiobis(2-nitrobenzoid acid) in the native enzyme with no impairment of catalytic activity. The rate of modification is unaffected by the presence of substrates. Reaction with N-ethylmaleimide leads to loss of catalytic activity, the rate of inactivation being increased by the presence of substrates. Peptides containing 361 amino-acid residues (about 88% of the total number in the protein) have been isolated and aligned by comparison with the known sequence of the isotopic isoenzyme from pig heart. In the regions compared, 342 of the residues are identical. Hence, assuming that those regions are representative of the whole, then the cytosolic isoenzymes from horse and from pig have about 95% identity of structure. Uniquely among the mammalian cytosolic aspartate aminotransferases so far examined, the enzyme from horse heart is acetylated at the N-terminus.

Uptake of aspartate aminotransferase into mitochondria in vitro causes efflux of malate dehydrogenase and vice versa.

Incubation of intact mitochondria with aspartate aminotransferase results in efflux of malate dehydrogenase and vice versa. The export process is specific and rapid. It shows saturation kinetics with respect to the effector enzyme consistent with involvement of a receptor for the effector in the mitochondrial membrane system. Export is inhibited by both beta-mercaptoethanol and by the metal chelating agent bathophenanthroline; both substances inhibit release of malate dehydrogenase by aspartate aminotransferase competitively whereas for release of aspartate aminotransferase by malate dehydrogenase inhibition is non-competitive. The efflux process is dependent on a trans-membrane pH gradient. Exported enzymes differ from the native forms in their dependence of activity on pH. Export of both aspartate aminotransferase and malate dehydrogenase is effected by incubation of mitochondria with the newly-synthesised precursor of aspartate aminotransferase; this observation provides supporting evidence for the physiological significance of the other results reported here. It is speculated that exported enzymes are on a pathway to degradation, and that coupled uptake and export is involved in the co-ordination of synthesis and breakdown of mitochondrial proteins.

Kinetic properties and thermal stabilities of mutant forms of mitochondrial aspartate aminotransferase.

Kinetic properties and thermal stabilities of the precursor form of mitochondrial aspartate aminotransferase, the mature form lacking 9 amino acids from the N-terminus, and forms of the mature protein in which cysteine-166 had been mutated to serine or alanine were compared with those of the mature enzyme. The precursor and the cysteine mutants showed moderately impaired catalytic properties consistent with decreased ability to undergo transition from the open to the closed conformation which is an integral part of the mechanism of action of the enzyme. The deletion mutant had a kcat only 2% of that of the mature enzyme but also much reduced Km values for both substrates. In addition it showed enhanced reactivity of cysteine-166 with 5,5'-dithiobis(2-nitrobenzoate), which is characteristic of the closed form of the enzyme, with no enhancement of reactivity in the presence of substrates. This is taken to show that the deletion mutant adopts a conformation that is significantly different from that of the mature enzyme particularly in respect of the small domain. The deletion mutant was found to be more resistant to thermal inactivation over a range of temperatures than were the other forms of the enzyme consistent with its having a more tightly packed small domain.

The primary structure of mitochondrial aspartate aminotransferase from human heart.

The complete amino acid sequence of the mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) from human heart has been determined based mainly on analysis of peptides obtained by digestion with trypsin and by chemical cleavage with cyanogen bromide. Comparison of the sequence with those of the isotopic isoenzymes from pig, rat and chicken showed 27, 29 and 55 differences, respectively, out of a total of 401 amino acid residues. Evidence for structural microheterogeneity at position 317 has also been obtained.

Transport of proteins into mitochondria.

There is still much that is obscure concerning the transport of proteins into or through the mitochondrial membrane systems. In addition, as pointed out previously, it is unlikely that the details of the process are the same for proteins destined for different compartments of the organelle. A brief summary of the process for matrix proteins might be as follows: The proteins are synthesized on free polysomes as precursors of higher molecular weight than the native forms. These precursors are liberated into the cell cytosol and subsequently translocated into the mitochondria. This timing might be different in yeast under some circumstances, synthesis being completed in association with the mitochondria. The precursors interact with a receptor in the outer mitochondrial membrane interaction being mediated by the presequences of the precursors. The presequences therefore act as addressing signals as well as possibly playing a role in one or all of (a) solubilization of precursors, (b) prevention of premature assembly into multimeric structures, or (c) maintenance of nonnative configurations required for transport. Interaction occurs with a second receptor, this time in the inner membrane of the mitochondria, interaction being with multiple sites in the polypeptide chain. Transport across the inner membrane then occurs, this transport depending on a transmembrane electrochemical gradient of which the proton component is the essential part. Transport is accompanied or followed by proteolysis of the prepiece, and formation of the native structure. While steps 1 and 2 of this sequence can be considered well established, the remaining steps are still poorly understood or purely hypothetical. Nevertheless, this sequence of events is consistent with known facts about the process and provides a framework for future investigations.

Crystal structure of Saccharomyces cerevisiae cytosolic aspartate aminotransferase.

The crystal structure of Saccharomyces cerevisiae cytoplasmic aspartate aminotransferase (EC 2.6.1.1) has been determined to 2.05 A resolution in the presence of the cofactor pyridoxal-5'-phosphate and the competitive inhibitor maleate. The structure was solved by the method of molecular replacement. The final value of the crystallographic R-factor after refinement was 23.1% with good geometry of the final model. The yeast cytoplasmic enzyme is a homodimer with two identical active sites containing residues from each subunit. It is found in the "closed" conformation with a bound maleate inhibitor in each active site. It shares the same three-dimensional fold and active site residues as the aspartate aminotransferases from Escherichia coli, chicken cytoplasm, and chicken mitochondria, although it shares less than 50% sequence identity with any of them. The availability of four similar enzyme structures from distant regions of the evolutionary tree provides a measure of tolerated changes that can arise during millions of years of evolution.

Lack of correlation between mRNA expression and enzymatic activity of the aspartate aminotransferase isoenzymes in various tissues of the rat.

Little is known about control of expression of basal levels of the aspartate aminotransferases which are ubiquitous 'house keeping' enzymes in vertebrates. We have measured both mRNA and activity levels for both isoenzymes in various rat tissues as a function of age. Patterns of mRNA expression for the two isoenzymes were similar in a particular tissue about differed widely between tissues. Surprisingly, there was no simple correlation between mRNA levels and specific activities of the enzyme products. We conclude that translation for mRNA for these two isoenzymes is subject to tissue-specific, and in some cases age-related, regulation.

The role of metal ions in the uptake of aspartate aminotransferase and malate dehydrogenase into isolated rat liver mitochondria in vitro.

To gain further insight into the mitochondrial receptor area which allows selective uptake of both purified aspartate aminotransferase and malate dehydrogenase into mitochondria, the inhibition of metal complexing agents such as bathophenanthroline and tiron on the uptake of both enzymes has been investigated. In view of the nature of the inhibition found, we propose the existence of metal ion(s) at or near the aspartate aminotransferase, but far from the malate dehydrogenase binding site.

Isolation and identification of some urinary inhibitors of calcium phosphate formation.

Normal urine has been examined for substances which inhibit formation of calcium phosphate. A separation scheme involving ultrafiltration, precipitation, electrophoresis and paper chromatography was devised to isolate these substances. Contrary to what has been suggested in the literature for many years, the urines examined did not contain a potent unidentified inhibitor. The major anionic inhibitors were citric acid, pyrophosphate and isocitric acid. These substances together with a small contribution from the cations appeared to account for most, if not all, of the inhibitory activity of urine.

Nucleotide sequence of a cDNA coding for mitochondrial fumarase from human liver.

The nucleotide sequence of a 1.46 kb cDNA, selected from a human liver library by the expression of fumarase antigenic determinants, was determined using the dideoxy chain termination method. The cDNA contained an open reading frame extending from the extreme 5'-base and coding for a protein with 468 amino acids. This protein, with the exception of an N-terminal methionine, was identified as mitochondrial fumarase. The protein showed a high degree of identity of structure with the fumarase from Bacillus subtilis (56.6%) and a fumarase from Escherichia coli (product of the fumC gene, 59.3%), and a lower degree of identity with the aspartase of E. coli (37.2%).

The primary structure of mitochondrial aspartate aminotransferase from pig heart: peptides obtained by cleavage with pepsin and with Staphylococcus aureus protease.

Results obtained after digestion of mitochondrial aspartate aminotransferase from pig heart with pepsin and with the protease from S. aureus are described. Peptic digestion produced a very complex mixture of peptides, which were purified and analyzed; structural information contained in these peptides covered nearly the entire molecule. Moreover, the lengths of some individual peptides and the peculiar self-overlapping found with families of peptides from adjacent regions were especially useful and interesting. Not all the possible peptides originating after digestion with S. aureus protease were isolated and examined. However, the high specificity of this protease and its usefulness for sequence studies were confirmed. In particular, the S. aureus peptides obtained were important for establishing the amidation state of glutamic acid/glutamine residues.

The primary structure of mitochondrial aspartate aminotransferase from pig heart: peptides obtained by cleavage with thermolysin and chymotrypsin.

The production, purification and analysis of peptide derived by digestion of mitochondrial aspartate aminotransferase with thermolysin and chymotrypsin are described. Despite the complexity of the peptide mixture obtained and the relative shortness of the fragments produced, these digests proved to be very useful for the completion of the primary structure determination of the enzyme. In fact, information for 87% of the total structure was contributed by thermolytic peptides, and for 89% by the chymotryptic ones. Moreover some of these peptides were essential for elucidating controversial points of the sequence.

The primary structure of mitochondrial aspartate aminotransferase from pig heart: peptides obtained by cleavage at basic residues.

Results obtained as part of a study of the primary structure of mitochondrial aspartate aminotransferase from pig heart are described. In particular, the S-aminoethylated protein was digested with trypsin and with the lysine specific protease from A. mellea. In the first case peptides contained 221 out of the total of 401 amino acid residues in the protein were obtained. By contrast the digest with A. mellea protease was not examined exhaustively and six peptides containing 49 amino acid residues were isolated. Digestion of the trifluoroacetylated and S-aminoethylated protein with A. mellea protease yielded a mixture of large fragments three of which, containing 89 amino acid residues, are described here. The combined results of these three digests yielded 66.6% of the total structure, concentrated mainly in the N-terminal half of the protein.