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The amino acid sequence of the coenzyme-binding site of serine transhydroxymethylase from rabbit liver has been determined. After reduction with NaBH4 and aminoethylation, a first sample of enzyme was digested with thermolysin and a single phosphopyridoxyl peptide was isolated. A second sample of similarly treated enzyme was digested with chymotrypsin and three phosphopyridoxyl peptides clearly originating from a unique coenzyme-binding site were isolated. Sequence analysis of these peptides indicate the following structure: Val-Val-Thr-Thr-Thr-His-Lys(Pxy)-Thr-Leu. Sequence homologies of the active site of various pyridoxalphosphate enzymes are discussed in terms of a possible catalytic role and of evolution of this class of proteins. [ABSTRACT FROM AUTHOR]

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, soIutions 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. [ABSTRACT FROM AUTHOR]

1. A primary structure is presented for mitochondrial aspartate aminotransferase from pig heart based on previously published results [Barra et al. (1977) FEBS Lett. 83, 241-244; Doonan et al. (1979) Ital. J. Biochem. 28, 441-455; Barra et al. (1979) Ital. J. Biochem. 28, 456-477, 478-490] and a small amount of extra information given here. 2. A detailed comparison is made between the primary structures of the cytosolic and mitochondrial isozymes. These are homologous proteins with 47% identity of primary structure and do not differ appreciably in overall polarity. 3. Hydrophobic chromatography of the isozymes using alkyl agaroses suggests that the cytosolic but not the mitochondrial isozyme has a hydrophobic patch or pocket capable of binding to the alkyl side chains of the affinity medium. 4. Evidence is presented to show that the cytosolic isozyme contains some basic amino acids whose ionizations are not expressed under native conditions. The mitochondrial isozyme does not show this effect. 5. Predictions of the secondary structures of the isozymes are presented based on four predictive models. Minimum predictions are derived on the basis of agreement of three out of four of the individual predictions. These aggregate predictions underestimate the amount of ordered secondary structure in both isozymes and suggest a relatively low degree of similarity between the isozymes. Individual predictive models, on the other hand, reveal greater similarities in secondary structures between the two isozymes. [ABSTRACT FROM AUTHOR]

Syringopeptin 25-A is a phytotoxic amphiphilic lipodepsipeptide containing 25 amino acid residues, produced by some isolates of the plant pathogenic bacterium Pseudomonas syringae pv. syringae. Previous papers have reported its covalent structure and some of its biological properties. Attention has now been directed to define its conformation in solution, a structural feature regarded as important for understanding its possible role in the bacterial colonization of host plants, and its toxic action on the plant cell. Here we report the stereochemistry of its amino acid components, the complete interpretation of the two-dimensional NMR spectra and NOE data, and finally the structure obtained by computer simulations applying distance geometry and molecular dynamics procedures. The confirmation of the syringopeptin 25-A in aqueous solution includes three different structural regions interrupted by rigid 2,3-dehydro-aminobutyric acid residues: a loop from residue 2 to 6, a helicoidal zone from 8 to 15, and the lactone ring from 18 to 25. The three dimentional structure of the lactone moiety is very similar to that of two previously studied bioactive lipodepsinonapeptides. Preliminary circular dichroism evidence of conformational variations in solution of trifluoroethanol, which stimulates a membrane-like environment, are also reported. [ABSTRACT FROM AUTHOR]

A copper, zinc superoxide dismutase, has been isolated from the marine turtle Caretta caretta and the complete amino acid sequence obtained. The sequence was determined by isolation and analysis of peptides obtained after cleavage of the carboxymethylated apoenzyme with trypsin or Staphylococcus aureus protease. Turtle superoxide dismutase consists of 166 amino acid residues, which represents the largest number to date for a cytosolic copper,zinc superoxide dismutase. The comparison of this amino acid sequence with that of bovine superoxide dismutase revealed a one-residue C-terminal extension, two single residue insertions and a 12-residue insertion in the N-terminal region, in turtle superoxide dismutase. The new segment consists of a threefold repeating sequence and was found to be the site for selective proteolytic attack by trypsin under native conditions. The biochemical characteristics, the spectroscopic and catalytic properties as well as the thermal stability and the resistance to irreversible denaturation, were carefully examined and were very similar to those of other superoxide dismutases. These results indicate that the presence of the new polypeptide segment does not affect the main folding of the chain and the quaternary structure, nor the functional parameters of turtle superoxide dismutase. The possibility that the new insert constitutes a loop excluded from the protein scaffold providing the framework of the active site is also discussed. [ABSTRACT FROM AUTHOR]

cDNA encoding human 4-aminobutyrate aminotransferase (aminobutyrate:2-oxoglutarate aminotransferase) was prepared by polymerase chain reaction using mRNA from human neuroblastoma cells as the template and oligonucleotides synthesized on the basis of the information obtained from direct protein sequencing. The cDNA-deduced sequence enabled peptides, sequenced by automated Edman degradation, to be aligned for confirmation of the complete primary structure. The results are compared with the recently published sequence of the rat enzyme deduced entirely from DNA sequencing [Medina-Kauwe, L. K., Tillakaratne, N. J. K., Wu. J.-Y. & Tobin, A, J, (1994) J. Neurochem. 62. 1267–1275]. Although the sequences are almost identical for most of their length, they differ in a segment of 36 residues. Almost complete identity o r the two sequences is established if it is assumed that a frame-shift error was introduced into the reported rat cDNA sequence. The human cDNA was used to probe for the presence of 4-aminobutyrate aminotransferase mRNA in human tissues and a significant transcript was found in heart, placenta and in tissues usually associated with the expression of this enzyme. [ABSTRACT FROM AUTHOR]

The amino acid sequence of pig liver 4-aminobutyrate aminotransferase has been determined by gas-phase sequencing of proteolytically derived peptide fragments. The sequence differs substantially from that predicted for the same enzyme on the basis of the sequence of cDNA derived from pig brain in recently published work [Kwon, O., Park, J. & Churchich, J. E. (1992) J. Biol. Chem. 267, 7215–7216]. Apart from a few minor differences, the two sequences are completely different in the segment of protein comprising the 36 residues at positions 107–142. Insertion of a cytosine between bases 402 and 403 in the cDNA sequence, together with deletion of the guanine at position 510, results in a DNA sequence which predicts exactly the amino acid sequence determined by peptide analysis in the present work. The mammalian enzyme has approximately 44% sequence identity with the same enzyme from two unicellular eukaryotes (Saccharomyces cerevisiae, Aspergillus nidulans) and 22% identity with that from Escherichia coli. [ABSTRACT FROM AUTHOR]

The amino acid sequence of glutamate decarboxylase from Escherichia coli was solved by a combination of automated Edman degradation of peptide fragments derived by proteolytic and chemical cleavage and sequencing of DNA. Correct alignment of three peptides, for which no peptide overlaps were available, was achieved by sequencing a 1.1-kbp fragment of DNA produced by a polymerase-chain reaction using primers corresponding to sequences known to be in amino-terminal and carboxy-terminal regions of the protein. Sequence similarity (24% identity) with mammalian glutamate decarboxylase was found to be limited to a 55-residue sequence around the lysine residue that binds the coenzyme. Stronger similarity (38% identity), again confined to the same region, is seen with bacterial pyridoxal-phosphate-dependent histidine decarboxylase. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of glutamate dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus has been determined. The sequence was reconstructed by automated sequence analysis of peptides obtained after cleavage by trypsin, cyanogen bromide, Staphylococcus aureus V8 protease and pepsin, The enzyme subunit is composed of 421 amino acid residues yielding a molecular mass of 46.078 kDa, The presence of N-ε,-methyllysine in six positions of the sequence was observed. Comparison of the sequence of glutamate dehydrogenase from S. solfataricus with the other known primary structures of the corresponding enzyme from different sources, gives an overall identity of 9.2% and shows a symmetrical evolutionary distance of this archaebactcrial protein from the two groups of vertebrate on one side and eubacterial and low eucaryote enzymes on the other side. The occurrence of specific substitutions and a possible role for N-ε-methylation of lysine residues are discussed in view of current hypotheses on the molecular basis of thermal adaptation of proteins. [ABSTRACT FROM AUTHOR]

Homogeneous preparations of cytosolic serine hydroxymethyltransferase from rabbit liver were incubated with several different proteases. Chymotrypsin rapidly cleaves a tetradecapeptide from the NH2-terminal end of the enzyme with the enzyme retaining full catalytic activity. Trypsin digestion results in the release of several small peptides from the NH2-terminal end of the enzyme. The remaining core protein is reduced in molecular mass by about 3500 Da. With L-serine as substrate the core protein has 1.5 times the activity of the native enzyme. The difference in activity is due to a change in Vmax since the Km values for L-serine and tetrahydrofolate are unchanged. When allothreonine is used as the substrate the activity of the trypsin-treated enzyme is unchanged. Ks values for glycine and several folate compounds are also unchanged for the trypsin-digested enzyme. The relative distribution of three glycine-enzyme complexes shows only small differences between the native and trypsin-digested enzyme. Thermal denaturation studies show that the trypsin-digested enzyme has a thermal transition three degrees lower than the native enzyme but the same enthalpy of denaturation. These results suggest that the 25-30 amino acid residues from the NH2-terminal end of the enzyme are not important in determining the catalytic activity and structural stability of the purified enzyme. Several other proteases had no observable effect on the activity and size of the enzyme. All of the proteases tested inactivated the apoenzyme and digested it into small fragments. The loss of enzyme activity in frozen liver is probably the result of the enzyme slowly being converted to the apoenzyme form, which is susceptible to protease degradation. [ABSTRACT FROM AUTHOR]

The inactivation of aspartate aminotransferase in the course of the incubation with L-serine O-sulfate is protected by thiosulfate. It has been found that also L-cysteine sulfinic acid is an inactivator when it is incubated with the enzyme, while L-serine-O-phosphate and L-cysteine-S-sulfonate are inactive. The inactivation by L-cysteine sulfinic acid is increased by the simultaneous addition of 2-oxoglutrate and is protected by thiosulfate. Cysteine-S-sulfonate has been detected in the incubates of the enzyme with L-serine-O-sulfate protected with thiosulfate. It has been concluded that the protection by thiosulfate is due to the β-addition of thiosulfate to the inactivator aminoacrylic acid. The resulting cysteine-S-sulfonate thus represents an inactive dead-end product formed in the course of the protection by thiosulfate. It is suggested that this reaction could play the role of protecting enzymes sensitive to aminoacrylic acid and of inserting inorganic sulfur into aminoacrylic acid, producing a cysteine derivative. [ABSTRACT FROM AUTHOR]

Both holo- and apo-aspartate-aminotransferase are sensitive to tetranitromethane; however, the inactivation which follows nitration is partial for the holoenzyme, and complete for the apoenzyme. Cysteinyl and tyrosyl residues are modified in both forms of the enzyme; in the apoenzyme a greater number of tyrosines are available to the reagent. The complete inactivation of the apoenzyme is correlated with the loss of only one tyrosyl residue, which is selectively modified under particular conditions. The loss of activity is caused by tile failure of the nitrated apoenzyme to recombine with the coenzyme. [ABSTRACT FROM AUTHOR]