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Biomedical subjects

N Ise

Publications and source records attributed to N Ise.

49 records · Page 3Linked to original sources
PubMed1982

Interaction of specific amino acid derivatives with dimeric alpha-chymotrypsin.

Binding and catalytic activities of dimeric alpha-chymotrypsin from specific amino acid derivatives were investigated with special reference to the equilibrium between active and inactive monomeric forms of this enzyme occurring in a low pH range. The low catalytic activity of the dimeric enzyme towards these specific substrates was revealed to be due to the difficulty in binding of the dimer. However the free tryptophanates and N-acetyl-L-tryptophan, which are slightly smaller (in molecular size) than the above substrates and comparable to the nonspecific phenyl acetate substrates (towards which the dimeric alpha-chymotrypsin showed an distinct catalytic activity in our previous study [J. Biochem. 87, 871-880 (1980)]), were bound to the dimer more strongly than to the monomeric enzyme. Hence they enhanced dimer formation when added at low concentrations.

Amino Acids↗
PubMed1982

pH dependence of the formation of dimeric alpha-chymotrypsin and its catalytic activity.

The dimerization of alpha-chymotrypsin (alpha-CT) has been known to involve a specific interaction between the Tyr-146 alpha-carboxyl of one molecule and the His-57 imidazole, which is a member of the catalytic triad in the active site, of the other. This interaction determines the pH dependences of the dimerization constant and of the catalytic activities of the monomeric and dimeric enzymes. We compared the pKa values for catalytic activities with known pKa values for the dimerization constant in order to assign pKa values to residues of the enzyme. In the monomeric enzyme, the catalytic triad has a pKa value of 3.6 at the site between the Asp-102 carboxyl and His-57 N delta 1, and the Tyr-146 alpha-carboxyl has a pKa value of 4.6. In the dimeric enzyme, the site between the Asp-102 carboxyl and His-57 N delta 1 has a pKa value of around 5.5 and the site between His-57 N epsilon 2 and the Tyr-146 alpha-carboxyl has a pKa value around 2.4. Protonation at the site between the Asp-102 carboxyl and His-57 N delta 1 reduced the catalytic activity of the dimeric enzyme for p-nitrophenyl propionate, indicating that the Asp-102 carboxyl plays an important role in the monomeric alpha-CT-catalyzed reactions.

Biopolymers↗
PubMed1982

pH and temperature dependences of thermolysin catalysis. Catalytic role of zinc-coordinated water.

The pH dependences of kcat/Km, kcat and Km of thermolysin-catalyzed hydrolysis of N-furylacryloylglycyl-L-leucinamide (Fua-Gly-LeuNH2) and N-furylacryloylglycyl-L-phenylalaninamide (Fua-Gly-PheNH2) were investigated. Taking the buffer dependences into account, the kcat/Km profile was explained by a simple bell-shaped curve with pKa1 = 5.0 and pKa2 = 8.25, at 25 degrees C. Both kcat and Km increased with pH at lower pH and took larger values for Fua-Gly-LeuNH2 than for Fua-Gly-PheNH2 at 25 degrees C. The pH dependence of inhibitory actions by amino acids and dipeptides, such as carbobenzyloxy-L-phenylalanine and L-phenylalanyl-L-leucinamide, showed characteristic features depending on their charge states: anionic or neutral ones inhibited the enzyme more strongly at lower pH while cationic ones did so at neutral pH. Temperature dependences of kcat/Km, Ki and the two pKa values in the kcat/Km profile were measured. The kcat/Km showed non-linear dependence while Ki increased linearly with temperature on a logarithmic scale. The calculated delta H values of deprotonation for pKa1 and pKa2 were 33.4 kJ/mol and 35.1 kJ/mol, respectively. The value for pKa1 is too large to be assigned to the carboxylic group of Glu-143, in contrast to the generally accepted view. A mechanism for thermolysin catalysis is presented with particular emphasis on the binding specificity and the catalytic role of zinc-coordinated water.

Catalysis↗