Hiroyuki Kagamiyama

Crystallization and properties of aspartate aminotransferase from escherichia coli B

Asparate aminotransferase (EC 2.6.1 .l) has been extensively studied as a representative of the pyridoxal 5’-phosphate (pyridoxal-P&dependent enzymes. The enzyme catalyzes the reversible transfer of the amino group between L-aspartic acid and cY-ketoglutaric acid, and plays an important role in nitrogen metabolism. Animal and plant tissues contain both mitochondrial and cytosolic isoenzymes [l] . The primary structures of both isoenzymes from pig heart muscle have been elucidated [2,3] . Preliminary crystallographic data have been reported for the large and single crystals of cytosolic [4,5] and mitochondrial isoenzymes [6]. Mammalian aspartate aminotransferases are immunochemically distinct from the bacterial enzymes [7] . Structural studies of the bacterial enzymes have not been done. The enzyme has been purified to homogeneity from Pseudomonas striata and crystallized [8] . However, its low content in cells and low yield in purification prompted a search for other bacterial sources to produce it more abundantly. The aspartate aminotransferases have been highly purified from Escherichia coli K-12 [9], its mutant [lo] and Crooks strain [ 1 l] , but none of them have been obtained in a crystalline form. We describe here high yield purification, crystallization and some properties of aspartate aminotransferase from Escherichia cob B as a first approach to the comparative studies on the enzyme.

The nucleotide sequence surrounding the promoter region of colicin E1 gene.

The nucleotide sequence of 570 bp, covering the N-terminal portion of the colicin E1 gene, was determined. The sequence of the N-terminal four amino acids of the colicin E1 protein, determined by manual Edman degradation, agreed with that predicted from the nucleotide sequence. From analysis of the 5-terminal sequences of RNAs synthesized in vitro, the promoter and operator regions of the colicin E1 gene were assigned. These data indicate the existence of two promoters, one of which is located in the coding region for colicin E1. DNA sequence homology of 16 bp was found between the putative operator regions of the colicin E1 and recA genes.

Cysteine sulfinate transamination activity of aspartate aminotransferases.

Abstract Aspartate aminotransferases from pig heart cytosol and mitochondria, Escherichia coli B and Pseudomonas striata accepted L-cysteine sulfinate as a good substrate. The mitochondrial isoenzyme and the Escherichia enzyme showed higher activity toward L-cysteine sulfinate than toward the natural substrates, L-glutamate and L-aspartate. The cytosolic isoenzyme catalyzed the L-cysteine sulfinate transamination at 50% the rate of L-glutamate transamination. The Pseudomonas enzyme had the same reactivity toward the three substrates. Antisera against the two isoenzymes and the Escherichia enzyme inactivated almost completely cysteine sulfinate transamination activity in the crude extracts of pig heart muscle and Escherichia coli B, respectively. These results indicate that cysteine sulfinate transamination is catalyzed by aspartate aminotransferase in these cells.

A sensitive method for the detection of aspartate: 2-oxoglutarate aminotransferase activity on polyacrylamide gels

Abstract A sensitive technique for the qualitative and semiquantitative determination of the activity of aspartate aminotransferase on polyacrylamide gels after electrophoresis is described. It relies on the ability of aspartate aminotransferase to produce SO3−− in the transamination between l -cysteine sulfinate and 2-oxoglutarate. The method is based on the reduction of nitroblue tetrazolium by SO3−− using phenazine methosulfate as a coupling agent. The method has been characterized using human and pig sera, crude homogenates and crystalline preparation from pig heart muscle, and bacterial crude extracts.

Nonidentical subunits of pyrocatechase from Pseudomonas arvilla C-1

Abstract Pyrocatechase [catechol:oxygen, 1,2-oxidoreductase (decyclizing), EC 1.13.11.1] from Pseudomonas arvilla C-1 has been reported to contain 2 g atoms of iron/mol of enzyme, based on a molecular weight of 90,000, determined by sedimentation and diffusion constants ( Y. Kojima, H. Fujisawa, A. Nakazawa, T. Nakazawa, F. Kanetsuna, H. Taniuchi, M. Nozaki, and O. Hayaishi, 1967 , J. Biol. Chem., 242, 3270–3278). The molecular weight was estimated again by sedimentation equilibrium and Sephadex G-200 gel filtration and found to be 63,000 and 60,000, respectively. The enzyme was also found to contain 1 g atom of iron/mol of enzyme, based on a molecular weight of 63,000. The enzyme was dissociated into two bands on polyarcylamide gel electrophoresis in the presence of either sodium dodecyl sulfate or 8 m urea, and was separated into two subunits, α and β, by CM-cellulose chromatography using a buffer solution containing 8 m urea. The molecular weights of the α and β subunits were determined to be 30,000 and 32,000, respectively, by sodium dodecyl sulfate-gel electrophoresis. The NH2-terminal sequences of these subunits determined by Edman degradation were as follows: α subunit, Thr-Val-Asn-Ile-Ser-His-Thr-Ala-Gln-Ile-Gln-Gln-Phe-Phe-Gln-Gln-(X)-(X)-Gly -Phe-Gly; β subunit, Thr-Val-Lys-Ile-Ser-His-Thr-Ala-Asp-Ile-Gln-Ala-Phe-Phe-Asn-Gln-Val-(X)-Gly-Leu-Asx. The COOH-terminal amino acid residues were determined to be alanine for the α subunit and glycine for the β subunit by three different methods: carboxypeptidase digestion, tritium labeling, and hydrazinolysis. These results indicate that the enzyme consists of two nonidentical subunits, α and β.

A new assay for l-aspartate: 2-oxoglutarate aminotransferase

Abstract A new enzymatic assay for aspartate aminotransferase is presented. The 2-oxoglutarate formed in transamination between l -glutamate and oxalacetate was determined in a system coupled with hydroxyglutarate dehydrogenase and NADH by following a decrease in absorbance at 340 nm. The method allowed accurate determination of the initial velocity of the reaction, which was proportional to the enzyme concentration. The Michaelis constants of pig heart cytosolic aspartate aminotransferase for l -glutamate and oxalacetate and the amino acceptor specificity using l -glutamate as an amino donor were determined. The method was applicable to the determination of the enzyme activity in various materials including rat serum and bacterial crude extract.

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: Proue5f8Alaue5f8Glnue5f8Aspue5f8Asnue5f8Serue5f8Argue5f8Pheue5f8Value5f8Ileue5f8Argue5f8Aspue5f8 Argue5f8Asnue5f8Trpue5f8Hisue5f8 Proue5f8Lysue5f8Alaue5f8Leuue5f8Thrue5f8Pro-Asp — Tyrue5f8Lysue5f8Thrue5f8Serue5f8Ileue5f8Alaue5f8Argue5f8 Serue5f8Proue5f8Argue5f8Glnue5f8Alaue5f8 Leuue5f8Value5f8Serue5f8Ileue5f8Proue5f8Glnue5f8Ser — Ileue5f8Serue5f8Gluue5f8Thrue5f8Thrue5f8Glyue5f8 Proue5f8Asnue5f8Pheue5f8Serue5f8Hisue5f8Leuue5f8 Glyue5f8Pheue5f8Glyue5f8Alaue5f8Hisue5f8Asp-His — Aspue5f8Leuue5f8Leuue5f8Leuue5f8Asnue5f8Pheue5f8Asnue5f8 Asnue5f8Glyue5f8Glyue5f8Leuue5f8 Proue5f8Ileue5f8Glyue5f8Gluue5f8Argue5f8Ile-Ile — Value5f8Alaue5f8Glyue5f8Argue5f8Value5f8Value5f8Aspue5f8 Glnue5f8Tyrue5f8Glyue5f8Lysue5f8Proue5f8 Value5f8Proue5f8Asnue5f8Thrue5f8Leuue5f8Value5f8Gluue5f8Met — Trpue5f8Glnue5f8Alaue5f8Asnue5f8Alaue5f8 Glyue5f8Glyue5f8Argue5f8Tyrue5f8Argue5f8 Hisue5f8Lysue5f8Asnue5f8Aspue5f8Argue5f8Tyrue5f8Leuue5f8Alaue5f8Pro — Leuue5f8Aspue5f8Proue5f8Asnue5f8 Pheue5f8Glyue5f8Glyue5f8Value5f8Glyue5f8 Argue5f8Cysue5f8Leuue5f8Thrue5f8Aspue5f8Serue5f8Aspue5f8Glyue5f8Tyrue5f8Tyr — Serue5f8Pheue5f8Argue5f8 Thrue5f8Ileue5f8Lysue5f8Proue5f8Glyue5f8Proue5f8 Tyrue5f8Proue5f8Trpue5f8Argue5f8Asnue5f8Glyue5f8Proue5f8Asnue5f8Asp — Trpue5f8Argue5f8Proue5f8Alaue5f8 Hisue5f8Ileue5f8Hisue5f8Pheue5f8Glyue5f8Ileue5f8 Serue5f8Glyue5f8Proue5f8Serue5f8Ileue5f8Alaue5f8Thr-Lys — Leuue5f8Ileue5f8Thrue5f8Glnue5f8Leuue5f8Tyrue5f8 Pheue5f8Gluue5f8Glyue5f8Aspue5f8Proue5f8 Leuue5f8Ileue5f8Proue5f8Metue5f8Cysue5f8Proue5f8Ileue5f8Val — Lysue5f8Serue5f8Ileue5f8Alaue5f8Asnue5f8 Proue5f8Gluue5f8Alaue5f8Value5f8Glnue5f8Glnue5f8 Leuue5f8Ileue5f8Alaue5f8Lysue5f8Leuue5f8Aspue5f8Metue5f8Asnue5f8Asn — Alaue5f8Asnue5f8Proue5f8Metue5f8 Asnue5f8Cysue5f8Leuue5f8Alaue5f8Tyrue5f8 Argue5f8Pheue5f8Aspue5f8Ileue5f8Value5f8Leuue5f8Argue5f8Glyue5f8Glnue5f8Argue5f8Lysue5f8Thrue5f8Hisue5f8 Pheue5f8Gluue5f8Asnue5f8Cys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.

Aspartate transaminase from E. coli: amino acid sequences of the NH2-terminal 33 residues and chymotryptic pyridoxyl tetrapeptide.

The amino acid sequences of pyridoxal-binding tetrapeptide and the NH2-terminal portion of aspartate transaminase from E.coli B were analyzed and compared with those of the corresponding parts of the cytosolic and mitochondrial isozymes from pig heart. After borohydride reduction and chymotryptic digestion of the E.coli enzyme, a pyridoxal-containing peptide was isolated, showing the sequence, Ser-Lys(Pxy)-Asn-Phe, identical with that of the cytosolic isozyme. The NH2-terminal sequence was determined up to 33 residues with a liquid phase sequence analyzer. Nearly the same degree of homology was observed among the NH2-terminal sequences of the three aspartate transaminases.

Pyridoxamine-α-keto acid transamination activity of aspartate apoaminotransferases

Abstract Pyridoxamine-α-keto acid transamination activities of homogeneous aspartate apoaminotransferases from various organisms were determined. Aspartate apoaminotransferases from pig heart cytosol and bakers yeast utilized both oxalacetate and α-keto-glutarate as amino acceptors, while those from pig heart mitochondria and bacteria ( Escherichia coli B and Pseudomonas striata ) showed reactivity only toward oxalacetate. Specific activities of bacterial aspartate apoaminotransferases were very high compared to those of the yeast and animal apoenzymes. Phosphate and various anions, including sulfate, raised the pyridoxamine-α-keto acid transamination activity of all the aspartate apoaminotransferases examined. However, a high concentration of phosphate inhibited the reaction.