Shigeru Kunugi

‘‘Ordered’’ structure in dilute solutions of sodium polystyrenesulfonates as studied by small‐angle x‐ray scattering

The small angle x‐ray scattering measurements were performed for aqueous solutions of sodium polystyrenesulfonates having relatively narrow molecular weight distributions. As was observed for other synthetic macroions, polynucleotide and proteins, a single, broad peak was observed. The scattering vector at the peak position (Sm) was shifted toward larger values with increasing polymer concentration and toward lower values with increasing salt concentration, which confirmed earlier observations with polyacrylate and poly‐L‐lysine. The molecular weight dependence of the scattering behavior, which was earlier observed, was confirmed to be true for samples with Mw of 74 000, 18 000, and 4600. The mixture of two fractions with different Mw’s gave a scattering curve which was again different from the composite curve obtained with the parent curves before mixing. A similar situation was observed for the mixture of polystyrenesulfonate and polyacrylate. Thus, it was concluded that the observed single peak indicat...

Pressure dependence of trypsin-catalyzed hydrolyses of specific substrates

The effects of pressure on the trypsin-catalyzed hydrolyzed hydrolyses of three specific substrates, N-benzoyl-L-arginine ethyl ester (BzArgOEt), amide (BzArgNH2) and p-nitroanilide (BzArgNA), have been examined. The volume of the activation (delta V++) for kcat was -2.4 ml/mol for BzArgOEt and +3 - +6 ml/mol for BzArgNH2. Because of different rate-determining steps in the steady-state kinetics, the delta V++ value for BzArgOEt would indicate the activation volume of the deacylation step, whereas that for BzArgNH2 the delta V++ for the acylation step. The activation volumes were accounted for in terms of the difference in the mechanisms on the formation and decomposition of the tetrahedral-like intermediates during the acylation and deacylation steps. The delta V values for the formation of BzArgNH2- and thionine-trypsin complexes were several ml/mol, consistent with the fact that the main driving force of the substrate binding to this enzyme is electrostatic interaction, and in contrast to the delta V values of alpha-chymotrypsin complex formation with indole (approximately 0 ml/mol) or 2-furylacryloyl-D-tryptophan methyl ester (approximately 0 ml/mol), for which the hydrophobic interaction is the dominant force of the substrate binding. For the hydrolysis of BzArgNA, which showed a distinct substrate activation at high substrate concentrations, the pressure dependence of the four parameters, ks, Ks, (the catalytic rate and dissociation constant of the normal enzyme-substrate complex, respectively), Kss and Kss (those of the complex activated by the binding of the second substrate molecule), were measured at 1 atm and 1000 atm (25 degrees C). All of the four parameters increased with increase in pressure.

‘‘Ordered’’ structure in dilute solutions of biopolymers as studied by small‐angle x‐ray scattering

Dilute aqueous solutions of bovine serum albumin, lysozyme, chondroitin sulfate, and tRNA were measured by small‐angle x‐ray scattering. The scattering curves showed a single, broad peak as was observed for synthetic polyelectrolytes, indicating the presence of an ordered distribution of charged solutes. The intermolecular distance evaluated from the peak position (2Dexpt) increased with decreasing polymer concentration and with increasing salt concentration. Except for chondroitin sulfate, 2Dexpt values were nearly equal to the interparticle distance (2D0) calculated based on the assumption of a uniform distribution. The observed relationship between 2Dexpt and 2D0 was in agreement with the proposal that intermacroion attraction is weak for low‐charge density particles under discussion. This attraction and repulsive interparticle interaction create a ‘‘secondary’’ minimum in the potential curve enabling ordering to take place. For tRNA, the scattering peak became lower with rising temperature. The fact t...

Study of the substituent effect on α-chymotrypsin-catalyzed hydrolysis of phenyl acetates by using a stopped-flow titration method

Abstract Many slow-reacting substrates for the pancreatic enzymes do not readily establish a steady state of the acylated enzyme. A novel method was used to measure their kinetic constants. After the enzyme and slow-reacting substrate had stood for a measured interval, a fast-reacting chromophoric substrate was added to determine (by its reaction velocity) the concentration of nonacylated enzyme. Substituent effects on the dissociation constant of enzyme-substrate complex (Ks), the second-order acylation rate constant ( k 2 K s ), and the first-order acylation rate constant (k2), as well as the parameters of the substrate activation of the deacylation step under the condition S0 ⪢ E0, have been investigated for various substituted phenyl acetates reacting with α-chymotrypsin. Although the σ− dependence of k 2 K s gave a ϱ value around 2, as had been reported in the literature, the Ks value showed a distinct σ− dependence. Taking into account the positive and negative steric effects of large p- and m-substituents, respectively, the ϱ value for the k2 step was found to be around 1. This small ϱ value indicates that the nucleophilic reaction occurs via the direct attack by Ser-195 Oγ on the carbonyl carbon of the substrate in these ester substrates, just as for the specific amino acid substrates, and that a nucleophilic attack of the His-57 imidazole nitrogen need not be postulated except in the case of nonspecific ester substrates.

Presteady-state kinetic study of the elementary processes in the chymotrypsin-catalyzed hydrolysis of specific ester substrate: Rate-limiting association process due to the secondary binding

Abstract Presteady-state kinetic studies of α-chymotrypsin-catalyzed hydrolysis of a specific chromophoric substrate, N-(2-furyl)acryloyl- l -tryptophan methyl ester, were performed by using a stopped-flow apparatus both under [E]0 ⪢ [S]0 and [S]0 ⪢ [E]0 conditions in the pH range of 5–9, at 25 °C. The results were accounted for in terms of the three-step mechanism involving enzyme-substrate complex (E · S) and acylated enzyme (ES′); no other intermediate was observed. This substrate was shown to react very efficiently, i.e., the maximum of the second-order acylation rate constant ( k 2 K s ′ ) max = 4.2 × 10 7 M −1 s −1 . The limiting values of Ks′ (dissociation constant of E · S), K2 (acylation rate) and k3 (deacylation rate) were obtained from the pH profiles of these parameters to be 0.6 ± 0.2 × 10−5 m, 360 ± 15 s−1 and 29.3 ± 0.8 s−1, respectively. Likewise small values were observed for Ki of N-(2-furyl)-acryloyl- l -tryptophan and N-(2-furyl)acryloyl- d -tryptophan methyl ester and Km of N-(2-furyl)acryloyl- l -tryptophan amide. The strong affinities observed may be due to intense interaction of β-(2-furyl)acryloyl group with a secondary binding site of the enzyme. This interaction led to a k −1 k 2 value lower than unity, i.e., the rate-limiting process of the acylation was the association, even with the relatively low k2 value of this methyl ester substrate, compared to those proposed for labile p-nitrophenyl esters.

Polyelectrolyte “Catalysis” on Complexation Reactions of Ni2+: Stopped-Flow and Concentration Jump Studies

The rate constants were measured for the complexation reaction oiNi2+ with several kinds of ligands in the presence of polyelectrolytes. In the reaction with an anionic ligand, the formation rate constant was decreased by the presence of either anionic or cationic polyelectrolyte, the dissociation rate constant being unchanged. The polyelectrolyte having hydrophobic residue enhanced the forward reaction with non-ionic ligands, as a result of a lowering of the enthalpy of activation. The backward rate constant was not affected. The equilibrium of the metal-ion binding by polyelectrolyte was discussed.