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K Tanizawa

Publications and source records attributed to K Tanizawa.

At least 73 records · Page 4Linked to original sources
PubMed1993

Site-directed random mutagenesis of AMP-binding residues in adenylate kinase.

Two highly conservative residues, Val67 and Gln101, in adenylate kinase are located in the hydrophobic region putatively involved in binding of the adenine ring of AMP. We have performed polymerase chain reaction-based random mutagenesis of the two residues using recombinant chicken muscle adenylate kinase cDNA as a template. The synthetic oligonucleotide primers contained A,G,C,T-mixed bases in the codons corresponding to those for Val67 and Gln101. The amplified fragments were ligated with the expression plasmid pKK223-3, and the mutant proteins expressed were identified by immunoblotting. Enzymatically active mutant proteins were selected on the basis of the growth at 45 degrees C of the temperature sensitive Escherichia coli mutant for adenylate kinase. At position 67, various amino acid residues other than Val have been found to restore the growth at 45 degrees C of the ts mutant. In contrast, only Gln, His, and Met could be present at position 101. These results are compatible with the proposal that Val67 contributes to the AMP binding through hydrophobic interactions and Gln101 by forming a hydrogen bond with the adenine ring. Indeed, several purified Val67 and Gln101 mutant enzymes exhibited markedly high Km values for AMP, whereas the Km values for MgATP were comparable to those of the wild-type enzyme. Substrate specificity for nucleoside monophosphates was changed significantly by the mutagenesis of the two residues.

Adenosine Monophosphate↗
PubMed1993

Exploring the active site in UDP-glucose pyrophosphorylase by affinity labelling and site-directed mutagenesis.

UDP-glucose pyrophosphorylase catalyses the reversible uridylyl transfer from UDP-glucose to MgPPi, forming glucose 1-phosphate and MgUTP. We have identified, by using uridine di- and tri-phosphopyridoxals, five lysyl residues located at or near the UDP-glucose-binding site. Another reactive analogue, pyridoxal diphosphoglucose, also modified the same set of lysyl residues. Based on the amounts of labels incorporated into each lysyl residue, we have provided a hypothetical model for possible locations of the five lysyl residues around the substrates bound to the enzyme. This model was consistent with the results of the mutagenesis studies. Lys367 is essential for the catalysis, whereas Lys263 may participate in the binding of PPi and/or glucose 1-phosphate in the binary complex. On the other hand, the results of affinity labelling with pyridoxal 5'-diphosphate suggest the role of Lys329 in the direct interaction with PPi in the enzyme-UDP-glucose complex.

Affinity Labels↗
PubMed1992

Anhydrosubtilisin-catalyzed peptide synthesis.

Anhydrosubtilisin was found to be a good catalyst for the peptide synthesis though its hydrolytic activity was fully inhibited. Amino acid (peptide) p-chlorophenyl ester (acyl donor) was coupled with amino acid (peptide) amide in the presence of the modified enzyme. The method did not afford by-products resulting from the hydrolysis of the acyl donor. This is the most advantageous characteristic of the method, since the hydrolysis of the acyl donor is unavoidable for the methods using catalytically active proteases. Anhydrosubtilisin was further shown to be useful for the fragment condensation of peptides.

Amidohydrolases↗
PubMed1992

Leucine dehydrogenase from Bacillus stearothermophilus: identification of active-site lysine by modification with pyridoxal phosphate.

We have constructed an efficient expression plasmid for the leucine dehydrogenase gene previously cloned from Bacillus stearothermophilus. The recombinant enzyme was overproduced in Escherichia coli cells to a level of more than 30% of the total soluble protein upon induction with isopropyl beta-D-thiogalactopyranoside. The enzyme could be readily purified to homogeneity by heat treatment and a single step of ion-exchange chromatography. The purified enzyme was inactivated in a time-dependent manner upon incubation with pyridoxal 5'-phosphate (PLP) followed by reduction with sodium borohydride. The inactivation was completely prevented in the copresence of L-leucine and NAD+. Concomitantly with the inactivation, several molecules of PLP were incorporated into each subunit of the hexameric enzyme. Sequence analysis of the fluorescent peptides isolated from a proteolytic digest of the modified protein revealed that Lys80, Lys91, Lys206, and Lys265 were labeled. Among these residues, Lys80 was predominantly labeled and, in the presence of L-leucine and NAD+, was specifically protected from the labeling. Furthermore, a linear relationship of about 1:1 was observed between the extent of inactivation and the amount of PLP incorporated into Lys80. A slightly active mutant enzyme, in which Lys80 is replaced by Ala, was not inactivated at all by incubation with PLP, showing that the inactivation is correlated with the labeling of only Lys80. Lys80is conserved in the corresponding regions of all the amino acid dehydrogenase sequences reported to date. These results suggest that Lys80 is located at the active site and plays an important role in the catalytic function of leucine dehydrogenase.

Amino Acid Oxidoreductases↗
PubMed1991

Potato tuber type H phosphorylase isozyme. Molecular cloning, nucleotide sequence, and expression of a full-length cDNA in Escherichia coli.

Higher plant tissues contain two alpha-glucan phosphorylase isozymes (EC 2.4.1.1), types L and H, localized in the plastid and the cytoplasm, respectively. We already isolated and sequenced a cDNA clone encoding the type L isozyme. Presently, a cDNA clone encoding the type H counterpart was isolated from a cDNA library of immature potato tuber by plaque hybridization, using two oligonucleotide probes synthesized based on the partial amino acid sequences of the type H isozyme. The message encodes a polypeptide of 838 amino acid residues. Sequence comparison of the two potato tuber phosphorylase isozymes revealed two major distinctions; the type L isozyme contains a 78-residue insertion in the middle of the polypeptide chain as well as a 50-residue amino-terminal extension. Except for these extra portions, the two isozyme sequences show an identity of 63%. The entire structural gene for the type H isozyme was inserted 3'-downstream of the strong T7 RNA polymerase promoter in the expression plasmid pET-3b. Escherichia coli BL21 (DE3) cells carrying this plasmid produced active phosphorylase upon induction with isopropyl-beta-D-thiogalactoside at 22 degrees C. The expression is entirely dependent on the temperature; the bacteria did not produce a detectable amount of the active enzyme at 37 degrees C. Addition of pyridoxine to the culture medium was effective for the enzyme production.

Amino Acid Sequence↗
PubMed1991

Expression in Escherichia coli of UDP-glucose pyrophosphorylase cDNA from potato tuber and functional assessment of the five lysyl residues located at the substrate-binding site.

The entire structural gene for potato tuber UDP-glucose pyrophosphorylase has been amplified from its cDNA by the polymerase chain reaction and inserted into the expression plasmid pTV118-N downstream from the lac promoter. Escherichia coli JM105 cells carrying thus constructed plasmid produced the enzyme to a level of about 5% of the total soluble protein upon induction with isopropyl beta-D-thiogalactopyranoside. The recombinant enzyme purified to homogeneity in two column chromatographic steps was structurally and catalytically identical with the enzyme purified from potato tuber except for the absence of an N-terminal-blocking acetyl group. To examine functional roles of the five lysyl residues that had been identified by affinity labeling studies to be located at or near the active site of the enzyme [Kazuta, Y., Omura, Y., Tagaya, M., Nakano, K., & Fukui, T. (1991) Biochemistry (preceding paper in this issue)], they were replaced individually by glutamine via site-directed mutagenesis. The Lys-367----Gln mutant enzyme was almost completely inactive, and the Lys-263----Gln mutant enzyme had significantly decreased Vmax values with perturbed Km values for pyrophosphate and alpha-D-glucose 1-phosphate. Lys-329----Gln also exhibited increased Km values for these substrates but exhibited Vmax values similar to those of the wild-type enzyme. The two mutant enzymes Lys-409----Gln and Lys-410----Gln showed catalytic properties almost identical with those of the wild-type enzyme. Thus, among the five lysyl residues, Lys-367 is essential for catalytic activity of the enzyme and Lys-263 and Lys-329 may participate in binding of pyrophosphate and/or alpha-D-glucose 1-phosphate.

Amino Acid Sequence↗
PubMed1991

Thermostable alanine racemase of Bacillus stearothermophilus. Construction and expression of active fragmentary enzyme.

Limited proteolysis studies on alanine racemase suggested that the enzyme subunit is composed of two domains (Galakatos, N. G., and Walsh, C. T. (1987) Biochemistry 26, 8475-8480). We have constructed a mutant gene that tandemly encodes the two polypeptides of the Bacillus stearothermophilus enzyme subunit cleaved at the position corresponding to the predicted hinge region. The mutant gene product purified was shown to be composed of two sets of the two polypeptide fragments and was immunologically identical to the wild-type enzyme. The mutant enzyme, i.e. the fragmentary alanine racemase, was active in both directions of the racemization of alanine. The maximum velocity (Vmax) was about half that of the wild-type enzyme, and the Km value was about double. Absorption and circular dichroism spectra of the fragmentary enzyme were similar to those of the wild-type enzyme. An attempt was made to separately express in Escherichia coli a single polypeptide corresponding to each domain, but no protein reactive with the antibody against the wild-type alanine racemase was produced. Therefore, it is suggested that the two polypeptide fragments can fold into an active structure only when they are co-translated and that they correspond to structural folding units in the parental polypeptide chain.

Alanine Racemase↗
PubMed1991

Role of leucine 66 in the asymmetric recognition of substrates in chicken muscle adenylate kinase.

Adenylate kinase has two distinct binding sites for nucleotide substrates, MgATP and AMP. To identify the location of the site that specifically interacts with the adenine ring of AMP, we have substituted Ala, Gly, Val, Gln, and Trp for Leu66 of the recombinant chicken muscle enzyme by site-directed mutagenesis. All the purified Leu66 mutant enzymes exhibited an essentially identical circular dichroism spectrum and had thermal stabilities similar to the wild-type enzyme. Steady state kinetic analysis showed that the Leu66 mutant enzymes have significantly decreased Vmax values and markedly large Km values only for AMP. These results show that the binding site for the adenine ring of AMP in adenylate kinase is presumably located close to Leu66, which is invariant in all the enzymes so far sequenced. Significant inhibition of activities of the mutant enzymes and quenching of the Trp66 fluorescence by substrates suggest that in some Leu66 mutant enzymes, MgATP also binds to the AMP-binding site. Thus, Leu66 of adenylate kinase might play a role in the asymmetric recognition of the adenine ring of AMP from that of MgATP. Furthermore, the hydrophobicity of the residue at position 66 appears to be important for the positive cooperativity of substrate binding.

Adenosine Monophosphate↗
PubMed1991

Effect of substitution of a lysyl residue that binds pyridoxal phosphate in thermostable D-amino acid aminotransferase by arginine and alanine.

Lys-145 of the thermostable D-amino acid aminotransferase, which binds pyridoxal phosphate, was replaced by Ala or Arg by site-directed mutagenesis. Both mutant enzymes were purified to homogeneity; their absorption spectra indicated that both mutant enzymes contained pyridoxal phosphate bound non-covalently. Even though the standard assay method did not indicate any activity with either mutant, addition of an amino donor, D-alanine, to the Arg-145 mutant enzyme led to a slow decrease in absorption at 392 nm with a concomitant increase in absorption at 333 nm. This result suggests that the enzyme was converted into the pyridoxamine phosphate form. The amount of pyruvate formed was almost equivalent to that of the reactive pyridoxal phosphate in the mutant enzyme. Thus, the Arg-145 mutant enzyme is able to catalyze slowly the half-reaction of transamination. Exogenous amines, such as methylamine, had no effect on the half-reaction with the Arg-145 mutant enzyme. In contrast, the Ala-145 mutant enzyme neither underwent the spectral change by addition of D-alanine nor catalyzed pyruvate formation, in the absence of added amine. However, the Ala-145 mutant enzyme catalyzed the half-reaction significantly in the presence of added amine. These findings suggest that a basic amino acid residue, such as lysine or arginine, is required at position 145 for catalysis of the half-reaction. The role of the exogenous amines differs with various active-site mutant enzymes.

Alanine↗
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

Thermostable alanine racemase of Bacillus stearothermophilus: subunit dissociation and unfolding.

The guanidine hydrochloride-induced subunit dissociation and unfolding of thermostable alanine racemase from Bacillus stearothermophilus have been studied by circular dichroism, fluorescence and absorption spectroscopies, and gel filtration. The overall process was found to be reversible: more than 75% of the original activity was recovered upon reduction of the denaturant concentration. In the range of 0.6 to 1.5 M guanidine hydrochloride, the dimeric enzyme was dissociated into a monomeric form, which was catalytically inactive. The monomeric enzyme appeared to bind the cofactor pyridoxal phosphate by a non-covalent linkage, although the native dimeric enzyme binds the cofactor through an aldimine Schiff base linkage. The monomer was mostly unfolded, with the transition occurring in the range of 1.8 to 2.2 M guanidine hydrochloride.

Alanine Racemase↗
PubMed1991

Comparative affinity labeling with reactive UDP-glucose analogues: possible locations of five lysyl residues around the substrate bound to potato tuber UDP-glucose pyrophosphorylase.

By using two reactive analogues of UDP-Glc, uridine di- and triphosphopyridoxals, we have recently probed the substrate-binding site in potato tuber UDP-Glc pyrophosphorylase [EC 2.7.7.9]. In this work, pyridoxal diphospho-alpha-D-glucose was used for the same purpose. This compound is also a reactive UDP-Glc analogue but having its reactive group on the opposite side of the pyrophosphate linkage to those of the above two compounds. The enzyme was rapidly inactivated when incubated with the compound at very low concentrations followed by reduction with sodium borohydride. The inactivation was almost completely prevented by UDP-Glc and UTP. Complete inactivation correspond to the incorporation of 1.0 mol of the reagent per mol of enzyme monomer. The label was found to be distributed in five lysyl residues (Lys-263, Lys-329, Lys-367, Lys-409, and Lys-40. All of these results were similar to those obtained previously with the other compounds, suggesting the presence of a cluster of five lysyl residues at or near the substrate-binding site of this enzyme. However, the incorporations of labels into each lysyl residue differed depending on the compounds used. The substrate retarded the incorporations in different manners. Based on the combined results of the present and previous studies, a hypothetical model is presented for the possible locations of the five lysyl residues around the substrate bound to the enzyme. This model is consistent with the kinetic properties of mutant enzymes in which the five lysyl residues were individually replaced by glutamine via site-directed mutagenesis.

Affinity Labels↗
PubMed1991

Characterization of the half and overall reactions catalyzed by L-lysine:2-oxoglutarate 6-aminotransferase.

Significant differences were found in the reaction rate, and the substrate and reaction specificities between the half reactions and the overall reactions catalyzed by L-lysine: 2-oxoglutarate 6-aminotransferase. The half reactions between an amino donor and the enzyme-bound pyridoxal 5'-phosphate, and also between an amino acceptor and the bound pyridoxamine 5'-phosphate followed first order reaction kinetics. The extrapolated first order rate constants and dissociation constants of the substrates were determined for the half reactions: lysine, 0.87 min-1 and 5.5 mM; glutamate, 1.1 min-1 and 10.5 mM; alanine, 0.66 min-1 and 6.6 mM; 6-aminohexanoate, 0.43 min-1 and 13.3 mM; and 2-oxoglutarate, 0.33 min-1 and 2.5 mM. As compared with the values reported for the overall reactions [Soda, K., Misono, H., & Yamamoto, T. (1968) Biochemistry 7, 4102-4109], the reactivity of the inherent substrates was lower by over 4 orders in the half reaction than that in the overall reaction, and the reactivity of alanine with the bound pyridoxal 5'-phosphate was reduced to 10% of that in the overall reaction. The substrate specificity in the half reaction was much lower than that in the overall reaction, which was re-examined in a reaction system containing the same concentration of the enzyme as that for the half reactions. Lysine 6-aminotransferase catalyzes the transfer of only the terminal amino group of lysine to 2-oxoglutarate in the overall reaction. However, in the half reaction, the 2-amino group as well as the terminal one was transferred to the bound pyridoxal 5'-phosphate. The ratio of reactivity of the 2-amino group to that of the 6-amino group was considerably influenced by the pH of the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1990

Substitution of glutamine for lysine at the pyridoxal phosphate binding site of bacterial D-amino acid transaminase. Effects of exogenous amines on the slow formation of intermediates.

In bacterial D-amino acid transaminase, Lys-145, which binds the coenzyme pyridoxal 5'-phosphate in Schiff base linkage, was changed to Gln-145 by site-directed mutagenesis (K145Q). The mutant enzyme had 0.015% the activity of the wild-type enzyme and was capable of forming a Schiff base with D-alanine; this external aldimine was formed over a period of minutes depending upon the D-alanine concentration. The transformation of the pyridoxal-5'-phosphate form of the enzyme to the pyridoxamine-5'-phosphate form (i.e. the half-reaction of transamination) occurred over a period of hours with this mutant enzyme. Thus, information on these two steps in the reaction and on the factors that influence them can readily be obtained with this mutant enzyme. In contrast, these reactions with the wild-type enzyme occur at much faster rates and are not easily studied separately. The mutant enzyme shows distinct preference for D- over L-alanine as substrates but it does so about 50-fold less effectively than the wild-type enzyme. Thus, Lys-145 probably acts in concert with the coenzyme and other functional side chain(s) to lead to efficient and stereochemically precise transamination in the wild-type enzyme. The addition of exogenous amines, ethanolamine or methyl amine, increased the rate of external aldimine formation with D-alanine and the mutant enzyme but the subsequent transformation to the pyridoxamine-5'-phosphate form of the enzyme was unaffected by exogenous amines. The wild-type enzyme displayed a large negative trough in the circular dichroic spectrum at 420 nm, which was practically absent in the mutant enzyme. However, addition of D-alanine to the mutant enzyme generated this negative Cotton effect (due to formation of the external aldimine with D-alanine). This circular dichroism band gradually collapsed in parallel with the transformation to the pyridoxamine-5'-phosphate enzyme. Further studies on this mutant enzyme, which displays the characteristics of the wild-type enzyme but at attenuated rates, may yield information on the factors controlling the stereochemistry of the reaction as well as on the catalytic steps of the transaminase pathway.

Base Sequence↗
PubMed1990

The role of Leu-190 in the function and stability of adenylate kinase.

To elucidate the role of C-terminal region of chicken adenylate kinase (a single polypeptide consisting of 193 amino acid residues) in the catalysis and stability of the enzyme, a series of mutant proteins truncated in the C-terminal region has been prepared by successive replacements of the sense codons by a termination codon via site-directed mutagenesis. Removal of the three C-terminal residues did not affect the apparent Michaelis constants (Km values) for AMP and ATP, although the Vmax values decreased gradually in parallel with the length of the polypeptide chain. A sudden increase in Km values for substrates, in particular for ATP, was observed on removal of one additional residue (Leu-190), the Vmax value also being less than one-half of that of the mutant enzyme with 3 residues shorter than the wild-type enzyme. These results suggest the importance of the highly conservative Leu-190. Therefore, we further prepared the mutant enzymes through replacement of Leu-190 by a variety of other amino acid residues. They all had substantially lower Vmax values and decreased thermostabilities. Their apparent Km values for ATP also changed, whereas those for AMP were affected to a lesser extent. The hydrophobicity of amino acid residues at position 190 was found to positively correlate with the specificity constants (kcat/Km values) for ATP and also with the thermostability of the enzyme. The fluorescence emission of the Trp-190 mutant enzyme was quenched by the addition of ATP. It is suggested that the C-terminal residues, particularly those around Leu-190, are present in a hydrophobic region which may be involved in binding of ATP.

Adenosine Monophosphate↗
PubMed1990

Alanine dehydrogenases from two Bacillus species with distinct thermostabilities: molecular cloning, DNA and protein sequence determination, and structural comparison with other NAD(P)(+)-dependent dehydrogenases.

The gene encoding alanine dehydrogenase (EC 1.4.1.1) from a mesophile, Bacillus sphaericus, was cloned, and its complete DNA sequence was determined. In addition, the same gene from a moderate thermophile, B. stearothermophilus, was analyzed in a similar manner. Large parts of the two translated amino acid sequences were confirmed by automated Edman degradation of tryptic peptide fragments. Each alanine dehydrogenase gene consists of a 1116-bp open reading frame and encodes 372 amino acid residues corresponding to the subunit (Mr = 39,500-40,000) of the hexameric enzyme. The similarity of amino acid sequence between the two alanine dehydrogenases with distinct thermostabilities is very high (greater than 70%). The nonidentical residues are clustered in a few regions with relatively short length, which may correlate with the difference in thermal stability of the enzymes. Homology search of the primary structures of both alanine dehydrogenases with those of other pyridine nucleotide-dependent oxidoreductases revealed significant sequence similarity in the regions containing the coenzyme binding domain. Interestingly, several catalytically important residues in lactate and malate dehydrogenases are conserved in the primary structure of alanine dehydrogenases at matched positions with similar mutual distances.

Alanine Dehydrogenase↗
PubMed1990

UDP-glucose pyrophosphorylase from potato tuber: cDNA cloning and sequencing.

We have isolated a cDNA encoding UDP-glucose pyrophosphorylase from a cDNA library of immature potato tuber using oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme. The cDNA clone contained a 1,758-base-pair insert including the complete message for UDP-glucose pyrophosphorylase with 1,431 base pairs. The amino acid sequence of the enzyme inferred from the nucleotide sequence consists of 477 amino acid residues. All the partial amino acid sequences determined protein-chemically [Nakano et al. (1989) J. Biochem. 106, 528-532] confirmed the primary structure of the enzyme. An N-terminal-blocked peptide was isolated from the proteolytic digest of the enzyme protein, and the blocking group was deduced to be an acetyl group by fast atom bombardment-mass spectrometry. On the basis of the predicted amino acid sequence (477 residues minus the N-terminal Met plus an acetyl group), the molecular weight of the enzyme monomer is calculated to be 51,783, which agrees well with the value determined by polyacrylamide gel electrophoresis. In the cDNA structure, the open-reading frame is preceded by a 125-base-pair noncoding region, which contains a sequence being homologous with the consensus sequence for plant genes, and is followed by a 174-base-pair noncoding sequence including a polyadenylation signal. Amino acid sequence comparisons revealed that the potato UDP-glucose pyrophosphorylase is homologous to the enzyme from slime mold, Dictyostelium discoideum, but not to ADP-glucose pyrophosphorylases from rice seed and Escherichia coli.

Amino Acid Sequence↗