Setsuko Nakagawa

Role of Asp102 in the enzymatic reaction of bovine β-trypsin: A molecular orbital study☆

Abstract Using the X-ray structure of the complex of bovine β-trypsin with the basic pancreatic trypsin inhibitor, the hydrogen-bond structure consisting of Ser195, His57 and Asp102 is clarified in relation to the mechanism of the enzymatic reaction from an ab initio quantum chemical point of view. Under the influence of the inhibitor, of the three hydrogen bonds involving Ser214, His57 and Ala56 around Asp102, and of the other ionic amino acid residues, Asp102 plays a significant role in lowering the barrier height of the proton transfer from Ser195 to His57 without accepting a proton from His57. The principal cause of the barrier height lowering is the electrostatic interaction.

Anisotropic contribution in multicenter polarizabilities and first hyperpolarizabilities. Ab initio MP2 calculations of acetylene, ethylene, ethane and benzene

Abstract Multicenter polarizabilities and first hyperpolarizabilities of acetylene, ethylene, ethane and benzene are evaluated by using ab initio MP2 polarized one-electron potentials for developing a reliable and simple polarizable potential. Combination of classical isotropic induced dipoles at atom centers with anisotropic induced dipoles along bonds is recommended for describing a strong and inhomogeneous field, which often encounters in molecular dynamics simulations of solutions including ions and biological systems.

Polarized one-electron potentials fitted by multicenter polarizabilities and hyperpolarizabilities. Ab initio SCF-CI calculation of water

Abstract The electrostatic potential is usually evaluated by putting a test charge at several points on an appropriate molecular surface. The polarization effect is an indispensable long-range interaction as well as the electrostatic interaction. To evaluate the polarization effect, it is necessary to relax the wavefunction under the perturbed Hamiltonian. In the present work the polarized molecular potentials based on the ab initio SCF-CI method have been fitted by multicenter polarizabilities and hyperpolarizabilities. The polarization energies and induced dipole moments calculated by using the fitted parameters have been significantly improved by including the first hyperpolarizabilities in the case of a water molecule.

Ab initio molecular orbital study on the effects of zinc ion, its ligands and ionic amino acid residues for proton transfer energetics between glu 270 and Zn co-ordinated water molecule in carboxypeptidase A

Abstract Thezinc-water-Glu 270 system was reported from the X-ray crystallographic study of native carboxypeptidase A(CPA) (Lipscomb et al., 1968). General base catalysis by the γ-carboxylate of Glu 270 was proposed for peptidase activity of CPA. The effects of zinc ion and its ligands (Glu 72, His 69-Asp 142, His 196) for proton transfer between Glu 270 and Zn co-ordinated water molecule in CPA were studied by the ab initio SCFLCAO-MO method. The results show that the proton transfer from the Zn co-ordinated water molecule to the γ-carboxylate of Glu 270 is greatly promoted by the Zn ion and, conversely, is greatly inhibited by its ligands. The facilitation effect of Zn ion and the inhibition effect of its ligands for the proton transfer were analysed by using the energy decomposition analysis. Moreover, calculations including all side chains of ionic amino acid residues and main chain residues in CPA as point fractional charges were performed. The results show that the proton transfer is affected by the ionic amino acid residues and is not affected by the main chain residues.

Protein modeling of human prorenin using the molecular dynamics methods

To study the activation-inactivation mechanism of the renin zymogen, prorenin, a tertiary structural model of human prorenin was constructed using computer graphics and molecular dynamics calculations, based on the pepsinogen structure. This prorenin model shows that the folded prosegment polypeptide can fit into the substrate binding cleft of the renin moiety. The three positively charged residues, Arg10, Arg15, and Arg20, in the prosegment make salt bridges with Asp225, Glu331, and Asp60, respectively, in renin. Arg43, which is in the processing site, forms salt bridges with the catalytic residues of Asp81 and Asp269. These ionic interactions between the prosegment and the renin may contribute to keeping the prorenin structure as an inactive form.

Role of catalytic residues in the formation of a tetrahedral adduct in the acylation reaction of bovine β-trypsin: A molecular orbital study☆

In the acylation reaction of serine proteases the effect of amino acid residues on the geometrical change of the catalytic site from Michaelis to tetrahedral state was studied by using ab initio molecular orbital calculations. Amino acid residues in the catalytic site and the peptide substrate were calculated as a quantum mechanical region, and all the other amino acid residues and the calcium ion were included in the calculation as the electrostatic effects. The effects of Asp102, Asp194, N-terminus and the oxyanion binding site are large. The oxyanion binding site directly stabilizes the tetrahedral substrate. Asp102 stabilizes the enzyme intermediate, interacting with the protonated His57 residue. In order to elucidate the roles of Asp102 and the oxyanion binding site, energy decomposition analyses were done for the intermolecular interactions. The contribution of Asp102 and the oxyanion binding site to the decrease of energy in the geometrical change is due to the electrostatic effect. The energies of the proton shuttle from Ser195 O gamma to the leaving group of the substrate were calculated for amide and ester substrate models.

Theoretical studies of pKa'S for organic compounds in aqueous solution

Abstract p K a differences between formic acid and acetic acid and between methanol and methanethiol were calculated as a sum of the acidity difference in the gas phase and the solvation free-energy difference. The ab initio molecular-orbital method with MP3/6-31+G * and/or MP4/6-31+ +G ** basis sets and the molecular-dynamics free-energy perturbation technique were applied to the studies of the acidity and solvation, respectively. The estimated value for formic acid and acetic acid was in good agreement with the experimental value within a few p K a units, but that for methanol and methanethiol was different from the experimental value by 4–6 p K a units.

Molecular orbital study of the effects of ionic amino acid residues on proton transfer energetics in the active site of carboxypeptidase a

Abstract As part of a study of the catalytic mechanism of carboxypeptidase A, two proton transfers in the system Glu 270 H 2 OZnHis 69-Asp 142 are treated with an ab initio MO method. Results show that the proton transfers are strongly affected by the environment. It is predicted that the proton between His 69 and Asp 142 is covalently bonded to His 69.

The pKa value of His 57-Asp 102 couple in the active site of bovine pancreatic β-trypsin: A molecular orbital study

Abstract The charge relay hypothesis generated a large number of theoretical and experimental studies that tested the ideas involved. Opinion based upon theoretical and experimental studies is divided on the prediction, although there are many experimental data which do not support the hypothesis. The essential feature is the proton transfer from the histidine imidazole to the aspartate. Thus, we have performed the detailed calculations of the proton transfer from His 57 to Asp 102 including the environment of the couple in protonated bovine pancreatic β-trypsin. The charge state of the His 57-Asp 102 couple is greatly influenced by the environment of the enzyme around it. In this paper, it is shown that the proton between His 57 and Asp 102 is covalently bonded to the His 57 imidazole in the protonated β-trypsin. Our MO calculations, which support the neutral-pK-histidine theory as the results, do not support the charge relay mechanism.

Characterization of N-Linked Oligosaccharides Attached to Human Renin Expressed in COS Cells

To study the role of N-linked oligosaccharides attached to human renin, we generated three kinds of glycosylation-deficient renins in which one or both of two putative N-glycosylation sites was eliminated by amino acid replacement using site-directed mutagenesis. Examination of the three mutant renins (Asn-5 to Ala, Asn-75 to Ala, and both Asn-5 and -75 to Ala) expressed in COS cells demonstrated that both putative sites were certainly glycosylated with heterologous N-linked oligosaccharides. Moreover, the oligosaccharide chain attached at Asn-5 was different from that attached at Asn-75 in its molecular size. In addition, the secreted amount of the three mutant renins were different from one another, although the mutant and wild-type renins had practically the same specific activity. Our results suggest that the N-linked oligosaccharides have no effect on the enzymatic activity, but play an important role in stable secretion of human renin.