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.
Convenient methods for the purification of the soluble aspartate aminotransferase from pig heart muscle are described. The enzyme obtained is homogeneous in the ultracentrifuge and to starch gel electrophoresis at about pH 9.0. At lower pH, as first described by Fasella et. al. [1], a number of electrophoretically distinct species can be detected. Some of the properties of these are reported. The currently accepted value of the molecular weight of the enzyme is incorrect: ultracentrifuge studies gave a value of 78,600 ± 2,400 (23 determinations). Gel filtration experiments led to a similar value. Titration of apoenzyme with cofactor gives values of the equivalent weight of the protein which vary with the enzymic specific activity. Extrapolation to the maximum specific activity leads to a value for the equivalent weight of about 40,000. The enzyme has, therefore, two cofactor binding sites.
The phospholipase A from the venom of the common European honey bee (Apis mellifica) has been completely purified. The final product (13 g from 700 g of crude venom) readily crystallizes and is homogeneous with respect to starch gel electrophoresis at pH 8.0, isoelectric focussing in polyacrylamide gel in the pH range 3–10, and sedimentation and diffusion analysis in the ultracentrifuge. Only one N‐terminal residue, isoleucine, can be detected either by the Edman or dansyl methods. Quantitative N‐terminal analysis and gel filtration on Sephadex G‐100 give values for the molecular weight of about 19000. Ultracentrifugation studies lead to a value of about 40000: in concentrated solution the molecule exists, therefore, as a dimer. The identity of the enzyme as a phospholipase of the A2 type has been confirmed since, with 1‐oleyl‐2‐isolauroyl phosphatidyl ethanolamine as substrate, isolauric acid is liberated in 100% yield whereas no oleic acid is released. A method for the assay of the enzyme based on continuous titrimetric estimation of hexanoic acid liberated from 1,2‐dihexanoyl lecithin has been used to study various aspects of the activity of the enzyme.
Carboxymethylated aspartate aminotransferase was digested with a proteinase claimed to be specific for lysine residues. Complete cleavage occurred at 12 of the 19 lysine residues in the protein, but at the remaining seven residues cleavage was either restricted or absent. In addition, cleavage was observed at three of the 26 arginine residues. These results are discussed with reference to the amino acid residues adjacent to points of complete or restricted cleavage. The complete primary structure of aspartate aminotransferase, based on these and other studies, is given. Evidence for the assignment of some acid and amide side chains has been deposited as Supplementary Publication SUP 50050 (11 pp.) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975) 145, 5. The evidence for the assignment of residue 366 was less conclusive than for the other acid and amide side chains and is, therefore, given in the main paper.
A method has been developed which allows isolation of 0.3 -0.5 g of mitochondrial aspartate aminotransferase in five days starting from 10 pig hearts; the method does not involve initial preparation of mitochondria. Mitochondrial malate dehydrogenase and the cytoplasmic aspartate aminotransferase may conveniently be recovered from side fractions. The product mitochondrial aspartate aminotransferase is homogeneous as judged by various electrophoretic techniques and by N-terminal analysis. Crystals of the enzyme have been obtained both from concentrated, essentially salt-free, solutions and from solutions of ammonium sulphate. The amino acid composition, N and C-terminal amino acid sequences and subunit molecular weight have been determined; these characteristic properties are compared with those of the cytoplasmic isozyme from the same source.Aspartate aminotransferase is one of a small group of enzymes the members of which exist in tissues of higher organisms in two distinct forms, one associated with the soluble fraction and the other with mitochondria [ l -31. The isozymes of aspartate aminotransferase differ from one another in chemical, physical, catalytic and immunochemical properties (for a review, see [4]). The primary structure of the cytoplasmic enzyme from pig heart has now been established [5,6] and it therefore seemed of interest to carry out similar studies on the mitochondrial form to establish whether the structures are related and if so, the degree of homology between them; this information is not available for any other pair of cytoplasmic and mitochondrial isozymes. Our preliminary results showed that the structures are indeed related [7] and this conclusion has been independently confirmed [8,9].Existing methods for isolation of the mitochondrial isozyme [4,10] started from preparations of intact mitochondria and were not suitable for large scale work. We have developed a new method, starting from whole-cell homogenates, which exploits the cationic properties of the mitochondrial isozyme and allows rapid isolation of the protein in quantities sufficient Ahbreviufion. Dansyl, 5-dimethylaminonaphthalene-l-sulphonyl.Enzymes. Aspartate aminotransferase (EC 2.6
1. A method was devised to allow determination of intramitochondrial aspartate amino-transferase activity in suspensions of intact mitochondria. 2. Addition of purified rat liver mitochondrial aspartate aminotransferase to suspensions of rat liver mitochondria caused an apparent increase in the intramitochondrial enzyme activity. No increase was observed when the mitochondria were preincubated with the purified cytoplasmic isoenzyme. 3. These results suggest that mitochondrial aspartate aminotransferase, but not the cytoplasmic isoenzyme, is able to pass from solution into the matrix of intact rat liver mitochondria in vitro. 4. This system may provide a model for studies of the little-understood processes by which cytoplasmically synthesized components are incorporated into mitochondria in vivo.
The crystal structure of Saccharomyces cerevisiae cytoplasmic aspartate aminotransferase (EC 2.6.1.1) has been determined to 2.05 w resolution in the presence of the cofactor pyridoxaL5'-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.Keywords: aspartate aminotransferase; crystal structure; maleate; pyridoxal phosphate Given that analyses of whole genome sequences depend on sequence similarity for assignment of biological function to open reading frames, structural studies of the same protein from many distantly related proteins are extremely important. Such studies help to define the level of similarity that can be tolerated by various folds with retention of function. They also serve to define the kinds of tertiary and secondary structural changes that occur at low levels of sequence identity.L-Aspartate aminotransferase ( L -A s~ AT, EC 2.6.1.1) is an excellent candidate for comparative studies of sequence and structure conservation. It is a key metabolic enzyme that links amino acid metabolism to carbohydrate metabolism through catalysis of the reversible transamination reaction.L-aspartate + 2-oxoglutarate w oxaloacetate + L-glutamate. Abbreviarionst PLP, pyridoxal-5'-phosphate; PMSF, phenylmethyl sulfonyl fluoride; PEG 4,000, polyethylene glycol with average molecular weight of 4,000; L-AsP-AT, r-aspartate aminotransferase. 3Current address:
1. The single (cytosolic) aspartate aminotransferase was purified in high yield from baker's yeast (Saccharomyces cerevisiae). 2. Amino-acid-sequence analysis was carried out by digestion of the protein with trypsin and with CNBr; some of the peptides produced were further subdigested with Staphylococcus aureus V8 proteinase or with pepsin. Peptides were sequenced by the dansyl-Edman method and/or by automated gas-phase methods. The amino acid sequence obtained was complete except for a probable gap of two residues as indicated by comparison with the structures of counterpart proteins in other species. 3. The N-terminus of the enzyme is blocked. Fast-atom-bombardment m.s. was used to identify the blocking group as an acetyl one. 4. Alignment of the sequence of the enzyme with those of vertebrate cytosolic and mitochondrial aspartate aminotransferases and with the enzyme from Escherichia coli showed that about 25% of residues are conserved between these distantly related forms. 5. Experimental details and confirmatory data for the results presented here are given in a Supplementary Publication (SUP 50164, 25 pages) that has been deposited at the British Library Document Supply Centre, Boston Spa. Wetherby, West Yorkshire LS23 7 BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5.
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