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H Kagamiyama

Publications and source records attributed to H Kagamiyama.

At least 91 records · Page 5Linked to original sources
PubMed1991

Expression of bovine lung prostaglandin F synthase in Escherichia coli.

The full-length bovine lung prostaglandin(PG) F synthase cDNA was constructed from partial cDNA clones and ligated into bacterial expression vector pUC8 to develop expression plasmid pUCPF1. This plasmid permitted the synthesis of bovine lung PGF synthase in Escherichia coli. The recombinant bacteria overproduced a 36-KDa protein that was recognized by anti-PGF synthase antibody, and the expressed protein was purified to apparent homogeneity. The expressed protein reduced not only carbonyl compounds including PGD2 and phenanthrenequinone but also PGH2; and the Km values for phenanthrenequinone, PGD2, and PGH2 of the expressed protein were 0.1, 100, and 8 microM, respectively, which are the same as those of the bovine lung PGF synthase. The protein produced PGF2 alpha from PGH2, and 9 alpha, 11 beta-PGF2 from PGD2 at different active sites. Moreover, the structure of the purified protein from Escherichia coli was essentially identical to that of the native enzyme in terms of C-terminal sequence, sulfhydryl groups, and CD spectra except that the nine amino acids provided by the lac Z' gene of the vector were fused to the N-terminus. These results indicate that the expressed protein is essentially identical to bovine lung PGF synthase. We confirmed that PGF synthase is a dual function enzyme catalyzing the reduction of PGH2 and PGD2 on a single enzyme and that it has one binding site for NADPH.

Amino Acid Sequence↗
PubMed1991

Purification and cDNA cloning of rat 6-pyruvoyl-tetrahydropterin synthase.

6-Pyruvoyl-tetrahydropterin synthase, which catalyzes the second step in the biosynthesis of tetrahydrobiopterin, was purified approximately 18,000-fold to apparent homogeneity from rat liver. The molecular mass of the native enzyme was estimated to be 83 kDa by gel filtration. The enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 17 kDa. Up to 24 residues of the NH2-terminal sequence were determined by Edman degradation, which released a single amino acid at each step. These results indicate that the enzyme consists of identical subunits. The purified enzyme was digested with lysyl endopeptidase or V8 protease, and 11 peptide fragments were isolated. On the basis of the sequences of these peptides, oligonucleotides were synthesized and used to screen a rat liver cDNA library, and one cDNA clone was isolated. The complete nucleotide sequence of the 1176-base pair cDNA was then determined. The deduced amino acid sequence contained 144 amino acid residues, but a NH2-terminal four-amino acid sequence was not found in the purified protein. Therefore, the mature protein consists of 140 amino acids. A single mRNA band of 1.3 kilobases was obtained by RNA blot analysis of rat liver. The predicted amino acid sequence of 6-pyruvoyl-tetrahydropterin synthase was compared with the Protein Sequence Database of the National Biomedical Research Foundation, revealing significant local similarity to large T antigens from the polyomavirus family.

Alcohol Oxidoreductases↗
PubMed1991

Site-directed mutagenesis of Escherichia coli aspartate aminotransferase: role of Tyr70 in the catalytic processes.

Site-directed mutagenesis of Tyr70 in the active site of Escherichia coli aspartate aminotransferase (AspAT) followed by kinetic studies has elucidated the roles of the hydroxyl group and benzene ring of Tyr70. X-ray crystallographic analysis showed that replacement of Tyr70 by Phe did not alter the active-site conformation of the enzyme. Comparison of the kinetic parameters of the four half-transamination reactions (the pyridoxal 5'-phosphate form of the enzyme with L-aspartate or L-glutamate and the pyridoxamine 5'-phosphate form with oxalacetate or 2-oxoglutarate) between the wild-type and [Tyr70----Phe]AspATs showed that the mutation increases the energy level of the transition state by 2 kcal.mol-1 for all the four substrates, suggesting some contribution of the hydroxyl group of Tyr70 to the transition state. When Phe70 was further replaced by Ser, the energy level of the transition state for L-glutamate or 2-oxoglutarate, but not for L-aspartate or oxalacetate, was further increased by 2-3 kcal.mol-1, suggesting that the presence of a benzene ring at position 70 is essential for recognizing the L-glutamate-2-oxoglutarate pair as substrates.

Amino Acid Sequence↗
PubMed1991

The role of His143 in the catalytic mechanism of Escherichia coli aspartate aminotransferase.

In aspartate aminotransferase (AspAT), His143 is located within a hydrogen-bonding distance to Asp222 that forms a strong ion pair with the ring nitrogen of the coenzyme, pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP). His143 of Escherichia coli AspAT was replaced by Ala or Asn. The mutant enzyme H143A showed a slight increase in the maximum velocity of the overall transamination reaction between aspartate and 2-oxoglutarate, while H143N AspAT showed a decrease to 60% in the maximum rate of the overall reactions in both directions. In all of the half-transamination reactions with four substrates, aspartate, glutamate, oxalacetate, and 2-oxoglutarate, the catalytic competence as defined by kmax/Kd decreased by 3-18-fold upon replacing His143 by either Ala or Asn. The extent of the decrease varied from one substrate to another; it was largely contributed to by the decrease in affinities for all substrates. The equilibrium constants, [PMP-form] [keto acid]/[( PLP-form] [amino acid]), decreased by over 10-fold upon the mutations at position 143. Both H143A and H143N AspATs exhibited a considerably decreased affinity for 2-methylaspartate, an external-aldimine-forming substrate analogue, yet without appreciable alteration in the affinity for succinate and glutarate, which are non-aldimine-forming analogues. All these findings suggest that, although His143 is not essential for catalysis, it might assist the formation of enzyme-substrate complex.

Aspartate Aminotransferases↗
PubMed1991

Localization of prostaglandin endoperoxide synthase in neurons and glia in monkey brain.

The localization of prostaglandin endoperoxide synthase in monkey brain was investigated by the immunoperoxidase method using the monoclonal antibody (PES-7) raised against the enzyme purified from bovine seminal vesicle. The frozen sections with 30-microns thickness were employed after the brain was fixed with perfusion of 2% paraformaldehyde in phosphate-buffered saline. The immunoreactivity was most intense in the neurons of cerebral cortex and hippocampus, and was moderate in the neurons of caudate nucleus, putamen, globus pallidus and amygdala, while it was relatively weak in glial cells in the whole brain regions including the white matter. The majority of neurons showed the immunoreactivity in the somata and proximal dendrites, but exceptionally in the pyramidal cells of the hippocampus, positive staining was also observed in the apical dendrites. In the cerebellum, the immunoreactivity in both neurons and glia was rather faint as compared with that in other regions. Positive staining was not significantly observed in the vasculatures and arachnoid membranes. These findings indicate that most of neuronal and glial cells in monkey brain contain the enzyme of the rate-limiting and initial step of the biosynthesis of prostaglandins which regulate a variety of neural functions.

Animals↗
PubMed1991

Thermostable aspartate aminotransferase from a thermophilic Bacillus species. Gene cloning, sequence determination, and preliminary x-ray characterization.

The gene encoding aspartate aminotransferase of a thermophilic Bacillus species, YM-2, has been cloned and expressed efficiently in Escherichia coli. The primary structure of the enzyme was deduced from nucleotide sequences of the gene and confirmed mostly by amino acid sequences of tryptic peptides. The gene consists of 1,176 base pairs encoding a protein of 392 amino acid residues; the molecular mass of the enzyme subunit is estimated to be 42,661 daltons. The active site lysyl residue that binds the coenzyme, pyridoxal phosphate, was identified as Lys-239. Comparison of the amino acid sequence with those of aspartate aminotransferases from other organisms revealed very low overall similarities (13-14%) except for the sequence of the extremely thermostable enzyme from Sulfolobus solfataricus (34%). Several amino acid residues conserved in all the compared sequences include those that have been reported to participate in binding of the coenzyme in three-dimensional structures of the vertebrate and E. coli enzymes. However, the strictly conserved arginyl residue that is essential for binding of the distal carboxyl group of substrates is not found in the corresponding region of the sequences of the thermostable enzymes from the Bacillus species and S. solfataricus. The Bacillus aspartate aminotransferase has been purified from the E. coli clone cell extracts on a large scale and crystallized in the buffered ammonium sulfate solution by the hanging drop method. The crystals are monoclinic with unit cell dimensions a = 121.2 A, b = 110.5 A, c = 81.8 A, and beta = 97.6 degrees, belonging to space group C2, and contain two molecules in the asymmetric unit. The crystals of the enzyme-alpha-methylaspartate complex are isomorphous with those without the substrate analog.

Amino Acid Sequence↗
PubMed1991

Cloning and sequencing of cDNA encoding rat GTP cyclohydrolase I. The first enzyme of the tetrahydrobiopterin biosynthetic pathway.

A full-length cDNA clone for GTP cyclohydrolase I, the first enzyme of the tetrahydrobiopterin biosynthetic pathway, was isolated and characterized. Synthetic oligonucleotides, constructed according to selected amino acid sequences of purified GTP cyclohydrolase I, were used to screen a rat liver cDNA library. Four clones were isolated, and the length of the longest cDNA insert was 1024 base pairs. The identity of the cDNA was confirmed by amino acid sequence data for eight fragments obtained by lysyl endopeptidase digestion of the purified protein. The coding region encoded a protein of 241 amino acid residues, but the NH2 terminus of the protein contained 11 additional amino acid residues not present in the purified protein. RNA blot analysis showed a single mRNA species of 1.2 kilobases in rat liver. A characteristic feature of the deduced amino acid sequence of GTP cyclohydrolase I was the presence of sequences similar to those proposed for the phosphorylation sites for casein kinase II and growth-associated histone H1 kinase. Furthermore, significant similarity was found to the highly conserved sequences of dihydrofolate reductases, which are known to be involved in the binding of the pterin group of dihydrofolate to the reductases. This region in GTP cyclohydrolase I may be assigned to the binding site of tetrahydrobiopterin, one of the inhibitors of this enzyme.

Amino Acid Sequence↗
PubMed1991

Tyr225 in aspartate aminotransferase: contribution of the hydrogen bond between Tyr225 and coenzyme to the catalytic reaction.

Tyr225 in the active site of Escherichia coli aspartate aminotransferase (AspAT) was replaced by phenylalanine or arginine by site-directed mutagenesis. X-ray crystallographic analysis of Y225F AspAT showed that the benzene ring of Phe225 was situated at the same position as the phenol ring of Tyr225 in wild-type AspAT. The mutations resulted in a great decrease in the rate of the transamination reaction, suggesting that Tyr225 is important for efficient catalysis. The kinetic analysis of half-transamination reactions of Y225F AspAT with four substrates (aspartate, glutamate, oxalacetate, and 2-oxoglutarate) and some analogues (2-methylaspartate, succinate, and glutarate) revealed a considerable increase in the affinities for all these compounds. In contrast, affinity for the amino acid substrates was decreased by mutation to arginine, but affinities for the keto acid substrates and the two dicarboxylates (succinate and glutarate) were increased. The electrostatic interaction between O(3') of the coenzyme [pyridoxal 5'-phosphate (PLP)] and the residue at position 225 affected the pKa value of the Schiff base, which is formed between the epsilon-amino group of Lys258 and the aldehyde group of PLP; based on the spectrophotometric titration the pKa values were determined to be 6.8 for wild-type AspAT, 8.5 for Y225F AspAT, and 6.1 for Y225R AspAT in the absence of substrate. The absorption spectra of the three AspATs were almost identical in the acidic pH region, but the spectrum of Y225F AspAT differed from that of wild-type or Y225R AspAT in the alkaline pH region.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Replacement of an interdomain residue Val39 of Escherichia coli aspartate aminotransferase affects the catalytic competence without altering the substrate specificity of the enzyme.

Three mutant Escherichia coli aspartate aminotransferases in which Val39 was changed to Ala, Leu, and Phe by site-directed mutagenesis were prepared and characterized. Among the three mutant and the wild-type enzymes, the Leu39 enzyme had the lowest Km values for dicarboxylic substrates. The Km values of the Ala39 enzyme for dicarboxylates were essentially the same as those of the wild-type (Val39) enzyme. These two mutant enzymes showed essentially the same kcat values for dicarboxylic substrates as did the wild-type enzyme. On the other hand, incorporation of a bulky side-chain at position 39 (Phe39 enzyme) decreased both the affinity (1/Km) and catalytic ability (kcat) toward dicarboxylic substrates. These results show that the position 39 residue is involved in the modulation of both the binding of dicarboxylic substrates to enzyme and the catalytic ability of the enzyme. Although the replacement of Val39 with other residues altered both the kcat and Km values toward various substrates including dicarboxylic and aromatic amino acids and the corresponding oxo acids, it did not alter the ratio of the kcat/Km value of the enzyme toward a dicarboxylic substrate to that for an aromatic substrate. The affinity for aromatic substrates was not affected by changing the residue at position 39. These data indicate that, although the side chain bulkiness of the residue at position 39 correlates well with the activity toward aromatic substrates in the sequence alignment of several aminotransferases [Seville, M., Vincent M.G., & Hahn, K. (1988) Biochemistry 27, 8344-8349], the residue does not seem to be involved in the recognition of aromatic substrates.

Aspartate Aminotransferases↗
PubMed1990

Purification and characterization of rho-crystallin from Japanese common bullfrog lens.

In a previous paper, we reported that the partial amino acid sequence (225 residues) from the COOH terminus of rho-crystallin from European common frog lens shows 77% similarity to that of prostaglandin (PG) F synthetase, an aldo-keto reductase, from bovine lung (Watanabe, K., Fujii, Y., Nakayama, K., Ohkubo, H., Kuramitsu, S., Kagamiyama, H., Nakanishi, S., and Hayaishi, O. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 11-15). Here rho-crystallin was purified to apparent homogeneity from the eye lens of the Japanese common bullfrog (Rana catesbeiana) by four sequential chromatographies using Sephadex G-100, Red Sepharose, and dual Mono S. Two types of rho-crystallin, RHO-I and RHO-II, named according to their elution order from a Mono S column, are essentially identical in terms of immunochemical properties, amino acid composition, and partial amino acid sequence. But the NH2-terminal Thr of RHO-I is blocked with an acyl group, while that of RHO-II is free. Both crystallins as well as PGF synthetase are monomeric proteins with a molecular weight of about 35,000 and they have the ability to bind NADPH with a stoichiometry of 0.75 mol of cofactor/mol of protein. Although rho-crystallin does not cross-react with antibody against PGF synthetase, the NH2-terminal amino acid sequence (107 residues) of rho-crystallin shows 77% similarity to that of the enzyme. However, PGD2, PGE2, 9,10-phenanthrenequinone, p-nitrobenzaldehyde, DL-glyceraldehyde, D-glucuronic acid, D-glucose, D-xylose, menadione, p-nitroacetophenone, dihydroxyacetone, succinic semialdehyde, phenylglyoxal, and testosterone were not substrates for these crystallins. PGH2 9,11-endoperoxide reductase activities of RHO-I and RHO-II were 1.3 and 1.0 milliunits/mg of protein, respectively, which are only about 2% of that of bovine lung PGF synthetase. These results indicate that the rho-crystallins RHO-I and RHO-II belong to a group of aldo-keto reductases based on primary structure, molecular properties, and NADPH-binding ability, but show only low PGH2 9,11-endoperoxide reductase activity.

Alcohol Dehydrogenase↗
PubMed1990

Pre-steady-state kinetics of Escherichia coli aspartate aminotransferase catalyzed reactions and thermodynamic aspects of its substrate specificity.

The four half-transamination reactions [the pyridoxal form of Escherichia coli aspartate aminotransferase (AspAT) with aspartate or glutamate and the pyridoxamine form of the enzyme with oxalacetate or 2-oxoglutarate] were followed in a stopped-flow spectrometer by monitoring the absorbance change at either 333 or 358 nm. The reaction progress curves in all cases gave fits to a monophasic exponential process. Kinetic analyses of these reactions showed that each half-reaction is composed of the following three processes: (1) the rapid binding of an amino acid substrate to the pyridoxal form of the enzyme; (2) the rapid binding of the corresponding keto acid to the pyridoxamine form of the enzyme; (3) the rate-determining interconversion between the two complexes. This mechanism was supported by the findings that the equilibrium constants for half- and overall-transamination reactions and the steady-state kinetic constants (Km and kcat) agreed well with the predicted values on the basis of the above mechanism using pre-steady-state kinetic parameters. The significant primary kinetic isotope effect observed in the reaction with deuterated amino acid suggests that the withdrawal of the alpha-proton of the substrates is rate determining. The pyridoxal form of E. coli AspAT reacted with a variety of amino acids as substrates. The Gibbs free energy difference between the transition state and the unbound state (unbound enzyme plus free substrate), as calculated from the pre-steady-state kinetic parameters, showed a linear relationship with the accessible surface area of amino acid substrate bearing an uncharged side chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartate Aminotransferases↗
PubMed1990

Effects of replacement of tryptophan-140 by phenylalanine or glycine on the function of Escherichia coli aspartate aminotransferase.

Trp140 of E. coli aspartate aminotransferase has been converted to Phe or Gly by site-directed mutagenesis. As compared to the wild-type enzyme, either of the mutant enzymes showed 10- to 100-fold increase in Km's for natural dicarboxylic substrates, but did not show appreciable changes in Km's for aromatic substrates. Teh kcat values for dicarboxylic and aromatic substrates were greatly decreased by [Trp140----Gly] mutation, but were decreased to lesser extents by [Trp140----Phe] mutation. These findings suggested that N(1) of Trp140 may not be essential for catalysis, but may be partly involved in the binding of the distal carboxylate group of the dicarboxylic substrates.

Aspartate Aminotransferases↗
PubMed1990

Three isozymes of catechol 1,2-dioxygenase (pyrocatechase), alpha alpha, alpha beta, and beta beta, from Pseudomonas arvilla C-1.

Three isozymes of catechol 1,2-dioxygenase (pyrocatechase) from Pseudomonas arvilla C-1 were separated using DEAE-Toyopearl chromatography. The specific activities of each isozyme were similar to one another. The molecular weights of isozymes 1, 2, and 3 were estimated to be approximately 67,000, 64,000, and 59,000, respectively, from gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isozymes 1 and 3 gave a single protein band, corresponding to Mr = 32,000 and 30,000, respectively, and isozyme 2 gave two bands corresponding to Mr = 32,000 and 30,000. These results indicated that isozymes 1 and 3 were homodimers, while isozyme 2 was a heterodimer. The NH2-terminal sequences up to 20 residues of these three isozymes confirmed that isozymes 1, 2, and 3 consisted of beta beta, alpha beta, and alpha alpha, respectively, based on our previous data (Nakai, C., Kagamiyama, H., Saeki, Y., and Nozaki, M. (1979) Arch. Biochem. Biophys. 195, 12-22). Properties of these isozymes such as absorption spectrum, iron content, substrate specificity, and kinetic constants were similar to one another. Subunit exchange between the different isozymes and dissociation of the isozymes into subunits was not observed under nondenaturing conditions. Available evidence indicates that these isozymes exist naturally in the bacterium and were not due to artifacts caused by purification.

Amino Acid Sequence↗
PubMed1990

Three-dimensional structures of aspartate aminotransferase from Escherichia coli and its mutant enzyme at 2.5 A resolution.

The structure of Escherichia coli aspartate aminotransferase complex with the inhibitor 2-methylaspartate, and that of the mutant enzyme in which an arginine was substituted for a lysine residue thereby forming a Schiff base with the coenzyme pyridoxal 5'-phosphate, were determined at 2.5 A resolution, by the molecular replacement method using the known structure of pig cytosolic aspartate aminotransferase. The enzyme catalyzes the reversible transamination between L-aspartate and alpha-ketoglutarate, and forms a dimeric structure of two identical subunits. Each subunit comprises two domains, a small and a large one. Although, in general, the overall and secondary structure of E. coli enzyme are similar to those of higher animals, some differences of enzymatic action between the enzyme from E. coli and those from higher animals could be explained on the basis of the X-ray structures and molecular mechanics calculation based on them.

Amino Acids↗
PubMed1990

Purification and characterization of thermostable aspartate aminotransferase from a thermophilic Bacillus species.

Aspartate aminotransferase (EC 2.6.1.1) was purified to homogeneity from cell extracts of a newly isolated thermophilic bacterium, Bacillus sp. strain YM-2. The enzyme consisted of two subunits identical in molecular weight (Mr, 42,000) and showed microheterogeneity, giving two bands with pIs of 4.1 and 4.5 upon isoelectric focusing. The enzyme contained 1 mol of pyridoxal 5'-phosphate per mol of subunit and exhibited maxima at about 360 and 415 nm in absorption and circular dichroism spectra. The intensities of the two bands were dependent on the buffer pH; at neutral or slightly alkaline pH, where the enzyme showed its maximum activity, the absorption peak at 360 nm was prominent. The enzyme was specific for L-aspartate and L-cysteine sulfinate as amino donors and alpha-ketoglutarate as an amino acceptor; the KmS were determined to be 3.0 mM for L-aspartate and 2.6 mM for alpha-ketoglutarate. The enzyme was most active at 70 degrees C and had a higher thermostability than the enzyme from Escherichia coli. The N-terminal amino acid sequence (24 residues) did not show any similarity with the sequences of mammalian and E. coli enzymes, but several residues were identical with those of the thermoacidophilic archaebacterial enzyme recently reported.

Amino Acid Sequence↗
PubMed1989

Purification and characterization of rat liver GTP cyclohydrolase I. Cooperative binding of GTP to the enzyme.

GTP cyclohydrolase I, an enzyme that catalyzes the first step in the biosynthetic pathway of tetrahydrobiopterin, has been purified about 38,000-fold to apparent homogeneity from rat liver extract with a yield of 5%. The molecular weight of the enzyme was estimated to be 300,000 by gel filtration on Ultrogel AcA 34. The purified enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at a position corresponding to a molecular weight of 30,000. N-terminal amino acid sequence analysis gave a single amino acid at every step of the Edman degradation up to residue 10. These results suggest that the enzyme is probably a homopolymer. The enzyme showed positive cooperativity with a Hill coefficient of 2.4 at a substrate (GTP) concentration of 10-50 microM. The Vmax value of the enzyme was 45 nmol/min.mg protein. The GTP concentration producing half-maximal velocity was 30 microM at a KCl concentration of 0.1 M. This value increased as the KCl concentration rose, without any change in Vmax or Hill number. Biosynthesis of tetrahydrobiopterin may be controlled by the intracellular level of GTP.

Aminohydrolases↗
PubMed1989

Substitution of a lysyl residue for arginine 386 of Escherichia coli aspartate aminotransferase.

Substitution of a lysyl residue for Arg-386 of Escherichia coli aspartate aminotransferase resulted in an extensive decrease in Vmax values (0.8% with the aspartate-2-oxoglutarate pair and 0.2% with the glutamate-oxalacetate pair, compared with the corresponding values for the wild-type enzyme). Kinetic analysis of the four sets of half-reactions, the pyridoxal form of the enzyme with aspartate or glutamate and the pyridoxamine form with 2-oxoglutarate or oxalacetate, allowed us to define the independent effect of the mutation on the reactivity of each substrate. Decrease in the first order rate constant (kmax) was more pronounced in the reactions with five-carbon substrates (glutamate and 2-oxoglutarate) than in those with four-carbon substrates (aspartate and oxalacetate), while the increase in the apparent dissociation constant (Kd) was greater for four-carbon substrates than for five-carbon substrates. The decrease of overall catalytic efficiency as judged by the values, kmax/Kd, was more pronounced in the reactions with five-carbon substrates than in those with four-carbon substrates. Affinities for substrate analogs such as succinate, glutarate, 2-methylaspartate, and erythro-3-hydroxyaspartate, were also considerably decreased by the mutation of the enzyme. These findings indicate that the side chain of the lysyl residue, although it bears a positive charge similar to that of the arginyl residue, is not structurally adequate for the productive binding of a substrate during catalysis.

Amino Acid Sequence↗