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K Tanizawa

Publications and source records attributed to K Tanizawa.

At least 145 records · Page 8Linked to original sources
PubMed1982

Comparative studies on the structure of active sites. Behavior of "inverse substrates" toward trypsin and related enzymes.

The kinetics of hydrolysis of "inverse substrates," p-amidinophenyl alkanoates, catalyzed by urokinase, plasmin, kallikrein, and trypsins from various sources were studied. Dissociation constants of acyl enzyme-ligand complexes, which are a characteristic parameter of the reaction with "inverse substrates," were analyzed with a view to comparing the spatial requirements of active sites. It was concluded that the spatial restraint of the active site as regards coexistence of the acyl residue and specific ligand is strictest for hog pancreatic kallikrein and this restraint decreases in the following order: human urokinase; bovine plasmin, bovine and hog trypsins; Streptomyces fradiae trypsin; and Streptomyces griseus trypsin.

Amidines↗
PubMed1982

Essential roles of alkylammonium and alkylguanidinium ions in trypsin-catalyzed hydrolysis of acetylglycine esters: enhancement of catalytic efficiency analyzed by the use of "inverse substrates".

An "inverse substrate" for trypsin, p-amidinophenyl acetylglycinate, has been synthesized and applied to mechanistic studies of tryptic hydrolysis. The compound reacts to form the intermediate acetylglycyl trypsin in a very specific manner, analogous to the reactions of p-amidinophenyl alkanoates with trypsin to give alkanoyl trypsins (Tanizawa, K., Kasaba, Y., & Kanaoka, Y. (1977) J. Am. Chem Soc. 99, 4485). The effects of alkylammonium and alkylguanidinium ions on tryptic hydrolysis of this "inverse substrate" have been investigated, and all alkylamines and alkylguanidines tested behaved as activators. n-Propylamine, n-propylguanidine, and n-butylguanidine, which have been reported to inhibit tryptic hydrolysis of ethyl or p-nitrophenyl acetylglycinate, were activators with p-amidinophenyl acetylglycinate. The activating properties of these amines and guanidines were demonstrated for the first time by making use of the specific characteristics of this inverse substrate. Tryptic mechanisms proposed in the literature are discussed in the light of these observations.

Catalysis↗
PubMed1982

Inverse substrates: novel synthetic substrates for trypsin and related enzymes.

The kinetics of the reactions of trypsin and related enzymes with esters derived from p-amidinophenol have been studied. These esters were named "inverse substrates" with respect to their structure and kinetic characteristics. Preparation of a variety of acyltrypsins was carried out by use of these substrates. Spectral analysis of acyl trypsins carrying a reporter group provided valuable information on the microenvironments near the active site. Kinetic analysis of the behaviour of trypsin and related enzymes toward these novel-type substrates revealed characteristic function of the enzymes not manifested with typical substrates. These results were analyzed with a view to comparing the spatial requirements of the respective active site.

Amidines↗
PubMed1981

Stereochemistry of meso-alpha,epsilon-diaminopimelate decarboxylase reaction: the first evidence for pyridoxal 5'-phosphate dependent decarboxylation with inversion of configuration.

The stereochemistry of the decarboxylation of meso-alpha,epsilon-diaminopimelate catalyzed by meso-alpha,epsilon-diaminopimelate decarboxylase (EC 4.1.1.20) of Bacillus sphaericus was determined by stereochemical analyses of [6-2H]-L-lysine produced by the reaction in D2O. The product [6-2H]-L-lysine was converted to levorotatory methyl 5-phthalimido[5-2H]valerate by the reactions not affecting the absolute configuration of the asymmetric carbon atom. By contrast, methyl 5-phthalimido[5-2H]valerate derived from [2,6-2H2]-L-lysine, which was produced from [2,6-2H2]diaminopimelate by decarboxylation in H2O, was dextrorotatory. The authentic methyl (R)-5-phthalimido[5-2H]valerate prepared from L-glutamate with glutamate decarboxylase was levorotatory. These results indicate that the meso-alpha,epsilon-diaminopimelate decarboxylase reaction proceeds in an inversion mode. The deuterium label in [6-2H]-L-lysine was fully conserved during the conversion into pelletierine through [1-2H]cadaverine by the stereospecific diamine oxidase reaction. Thus, the enzymatic decarboxylation of meso-alpha,epsilon-diaminopimelate occurs with inversion of configuration in contrast to the other amino acid decarboxylase reported so far.

Bacillus↗
PubMed1981

Analysis of latent properties of trypsin. Acyl trypsins derived from enantiomeric pairs of "inverse substrates".

p-Amidinophenyl esters derived from a variety of amino acids and peptides, including D-amino acids, were synthesized. The kinetic behavior of trypsin towards these esters, which are "inverse substrates," was analyzed. Deacylation rates of acyl trypsins carrying D-amino acid residues were determined for the first time by the use of these "inverse substrates." The steric requirements of the catalytic site region of trypsin were successfully analyzed by studying the deacylation process as manifested in the hydrolyses of enatiomeric pairs of "inverse substrates." The effects of chiral ligands on the deacylation process were also studied in connection with the chiral requirements of the active site.

Acylation↗
PubMed1981

Properties of crystalline L-ornithine: alpha-ketoglutarate delta-aminotransferase from Bacillus sphaericus.

The distribution of bacterial L-ornithine: alpha-ketoglutarate delta-aminotransferase (L-ornithine:2-oxo-acid aminotransferase [EC 2.6.1.13]) was investigated, and Bacillus sphaericus (IFO 3525) was found to have the highest activity of the enzyme, which was inducibly formed by addition of L-ornithine or L-arginine to the medium. L-Ornithine:alpha-ketoglutarate delta-aminotransferase, purified to homogeneity and crystallized from B. sphaericus, had a molecular weight of about 80,000 and consisted of two subunits identical in molecular weight (41,000) and in amino-terminal residue (threonine). The enzyme exhibited absorption maxima at 278,343, and 425 nm and contained 1 mol of pyridoxal 5'-phosphate per mol of enzyme. The formyl group of pyridoxal 5'-phosphate was bound through an aldimine linkage to the epsilon-amino group of a lysine residue of the protein. The enzyme-bound pyridoxal 5'-phosphate, absorbing at 425 nm, was released by incubation with phenylhydrazine to yield the catalytically inactive form. The inactive enzyme, which was reactivated by addition of pyridoxal 5'-phosphate, still had a 343-nm peak and contained 1 mol of a vitamin B6 compound. The holoenzyme showed positive circular dichroic bands at 340 and 425 nm, whereas the inactive form had no band at 425 nm. The enzyme was highly specific for L-ornithine and alpha-ketoglutarate and catalyzed delta-transamination between them to produce L-glutamate and L-glutamate-gamma-semialdehyde, which as spontaneously converted to delta 1-pyrroline-5-carboxylate. The enzyme activity was significantly affected by nonsubstrate amino acids, amines, and carbonyl reagents.

Amino Acids↗
PubMed1981

Behavior of trypsin and related enzymes toward amidinophenyl esters.

Kinetics of the hydrolysis of amidinophenyl esters catalyzed by urokinase, kallikrein, plasmin and trypsins from various origins were studied. P-Amidinophenyl esters in which the arrangement of the specific group for bovine trypsin (charged amidinium) is reversed to that of normal substrates were characterized as specific substrates for the enzyme and named "inverse substrates" (K. Tanizawa, Y. Kasaba and Y. Kanaoka, J. Am. Chem. Sc., 99, 4485-4488) Behavior of these enzymes toward amidinophenyl esters was demonstrated to be very similar to that of bovine trypsin. These observations account for the structural similarity of their active sites. p- Amidinophenyl esters were found to be hydrolyzed also by clostripain which is known as a thiol protease with trypsin-like specificity. This is the first example of the "inverse substrates" for a thiol enzyme.

Acylation↗
PubMed1980

Specific spin-labeling at trypsin active site. Application of 'inverse substrate' to the structural analysis of the active site.

Specific and reversible spin-labeling of trypsin (EC 3.4.21.4) active site was carried out by 'inverse substrate', 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl and 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolinyloxyl-p-amidinophenyl esters. The paramagnetic resonance spectra of the labeled trypsin in the form of acyl enzyme intermediate were investigated. The 2T value, separation of the outer hyperfine extrema, was observed to be sensitive to pH of the medium. These results are discussed in terms of pH-dependent conformational change at the vicinity of active site.

Acylation↗
PubMed1980

'Inverse' substrates for butyrylcholinesterase.

1. 'Inverse'-type substrates for butyrylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8), i.e., p- and o-nitrophenyl esters of (3-carboxypropyl)-trimethylammonium iodide, (4-carboxybutyl)trimethylammonium iodide and (5-carboxypentyl)trimethylammonium iodide were prepared, and their kinetic parameters for butyrylcholinesterase-catalyzed hydrolysis were determined. 2. The hydrolysis of these 'inverse'-type substrates were found to proceed through specific binding with the enzyme and efficient production of acyl enzyme intermediates, a pathway essentially identical with that followed by choline esters, normal type substrates.

Acetylcholinesterase↗
PubMed1980

Induced activation in the deacylation step of tryptic hydrolysis. An application of "inverse substrates" to mechanistic studies of the enzyme.

Trypsin [EC 3.4.21.4]-catalyzed hydrolysis of "inverse substrates" was investigated kinetically. "Inverse substrates" for trypsin are specific substrates in which the arrangement of the site-specific group is reversed compared to that of the normal substrate, e.g., a cationic center is included in the leaving group instead of being in the acyl moiety (Tanizawa, K., Kasaba, Y., & Kanaoka, Y. (1977) J. Am. Chem. Soc. 99, 4485-4488). Acyl enzyme intermediates formed specifically from these substrates are advantageous for the mechanistic analysis of trypsin action, since the cationic group liberated from the acyl moiety can no longer exhibit specific interaction with the enzyme binding site in the subsequent deacylation stage. Remarkable rate acceleration at the deacylation step was observed on adding amidinium or ammonium compounds. The effects of the size of the acyl moiety and the charged molecule on the acceleration were examined. Latent properties of 1-butylamine as an activator were found in the present study. Based on these observations, it is suggested that a cationic molecule which can be well accommodated together with the acyl group within the active center cleft causes rate enhancement, with associated conformational changes.

Acylation↗
PubMed1980

A novel chiral microenvironmental probe at the active site of trypsin. Extrinsic cotton effects of acyl-trypsin possessing an enantiomeric pair of chromophores.

p-Amidinophenyl esters of an enantiomeric pair of N-(2,4-dinitrophenyl)alanine (N2Ph-Ala) were both efficiently hydrolyzed by trypsin. The acylation and deacylation rate constants for the D-isomer are 1/2.5 of those for the L-isomer. Slow rates of deacylation of the two substrates made it possible to prepare the pair of enantiomeric acyl-trypsins. Circular dichroic (CD) spectra of the purified acyl-trypsins revealed that the two extrinsic chromophores are somewhat differently oriented in the chiral environment of the active site, although both chromophores could couple intermolecularly with similar intrinsic chromophores near the active site. When p-amidinophenol was added, not only was the deacylation rate of N2Ph-DAla-trypsin noticeably increased, but also the transient CD spectrum of the enzyme derivative changed markedly in comparison with that of the L-derivative. The observations indicate that the two enantiomeric acyl groups at the active site are situated in different microenvironments.

Acylation↗
PubMed1980

"Inverse substrates" for trypsin-like enzymes.

"Inverse substrates" for bovine thrombin and human plasmin were demonstrated. "Inverse substrates" for the enzymes are characterized as specific substrates in which the arrangement of site-specific group is reversed compared to that of normal substrate, e.g., a cationic center is included in their leaving group instead of being in their acyl moiety (K.Tanizawa, Y.Kasaba, Y.Kanaoka, J. Am. Chem. Soc. 99. 4485-4488). Kinetic characteristics of thrombin, plasmin and trypsin toward "inverse substrates" were compared. Based on these observations, differences in active centers of the trypsin homologs were discussed. Behavior of p- and m-hydroxyphenylguanidine derivatives as new "inverse substrates" for trypsin was also reported.

Animals↗
PubMed1980

New chromogenic and fluorogenic substrates for the determination of butyrylcholinesterase and arylesterase activities.

A simple direct spectrophotometric method for the determination of butyrylcholinesterase (EC 3.1.1.8) and arylesterase (EC 3.1.1.2) activities has been developed. New chromogenic substrates, (3-carboxypropyl)trimethylammonium iodide o-nitrophenyl ester (I) and (3-carboxypropyl)trimethylammonium iodide p-nitrophenyl ester (II), as well as new fluorogenic substrate, (3-carboxypropyl)trimethylammonium iodide 4'-methylumbelliferyl ester (III), were used in this study. Horse serum butyrylcholinesterase equally catalyzed hydrolysis of the compounds, I, II and III. Hydrolysis of these compounds by trypsin, chymotrypsin, acetylcholinesterase and carboxylesterase was negligible or quite slow. By human serum butyrylcholinesterase, however, only the compound I was preferentially hydrolyzed. The compound III, by contrast, was found to be a specific substrate for arylesterase of human serum without being affected by the butyrylcholinesterase. All these measurements were carried out readily and efficiently, by analyzing highly colored products with I and II, and highly fluorescent product with III.

Animals↗
PubMed1980

Tryptic hydrolysis of omega-amidino alpha-amino acid (indospecine) esters.

Several esters of a series of omega-amidino alpha-amino acids were synthesized. Some of them were shown to be efficiently hydrolyzed by bovine trypsin [EC 3.4.21.4]. The kinetics of hydrolysis of the esters were compared with those of the omega-guanidino series. The relationships between the length of the side chain and the susceptibility to hydrolysis by trypsin were somewhat different in the two series. These slight differences are discussed in terms of the molecular structures and their relation to the structural requirements of the trypsin active site.

Amino Acids, Diamino↗