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Electrostatic potentials for several enzymes and their guest molecules such as ligands and/or cofactors are individually calculated and illustrated as colour codes on their tertiary structures using a raster graphics technique. Two kinds of potential surface were illustrated on the molecular surface of the guest molecule. One is due to the host enzyme (H-on-G potential surface) and the other is due to the guest molecule itself (G-on-G potential surface). Remarkable mutual complementarities between the H-on-G and G-on-G potential surfaces were found in most cases. These give a semi-quantitative indication of the important role of electrostatic recognition in enzymes.

The heat of complex formation of H3PBH3 was calculated to be -15.3 kcal/mol by double zeta ab initio LCAO MO SCF calculations ; this is very similar to the experimental values for (CH3)3PB(CH3)3 and (CH3)3PBF3. The origin of complex formation of H3PBH3 was elucidated by energy decomposition methods. The order of contributions is ES-(41%)>CT (37%)> PL (22%). The d atomic orbitals on phosphorus play a role in increasing the polarization energy upon complex formation. The barrier to internal rotation of H3PBH3 was calculated to be 2.4 kcal/mol, which is in very good agreement with the experimental value of 2.47kcal/mol. The exchange repulsion and the charge transfer energy related to the staggered form contribute to the barrier to internal rotation. The change of the charge transfer energy corresponds to the difference of the barrier heights between H3PBH3 and H3SiCH3. The energies of complex formation of F3PBH3 and (CH3)3PBH3 were calculated, to investigate the origin of the barrier to internal rotation.

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 Oγ to the leaving group of the substrate were calculated for amide and ester substrate models.

Simulation of the enzymatic reaction of dogfish M4 lactate dehydrogenase (LDH) was performed. In the enzymatic reaction from L-lactate to pyruvate, the structure in which the proton transfer from L-lactate to His 195 is coupled with the hydride transfer from L-lactate to NAD+ was simulated. In the reverse reaction, the structure in which the proton transfer from His 195 to pyruvate is coupled with the hydride transfer from NADH to pyruvate was also simulated. Since the proton of His 195 approaches the carbonyl oxygen of pyruvate from the π direction of the pyruvate plane in the simulation, the electronic structure and the reactivity of pyruvate were studied from a quantum chemical point of view, using the ab initio LCAO MO method. When the carbonyl carbon of pyruvate is attacked by the hydride ion of NADH, the total π electron density of the carbonyl oxygen of pyruvate increases substantially. Therefore, the proton of His 195 attacks the carbonyl oxygen of pyruvate from the π direction. Moreover, the pyruvate-Arg 171 complex having two nearly parallel hydrogen bonds is more easily attacked by hydride than complexes having other conformations.

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.

In the enzymatic reaction of β-trypsin the role of environment around the catalytic triad is studied by means of ab initio molecular orbital calculations. The triple ion form of the catalytic triad (Asp 102(−)-His 57(+)-Ser 195(−)) is considerably more stable than the double proton-transferred form (Asp 102(neutral)-His 57(neutral)-Ser 195(−)), due to the environment around it, rather than its nature. The “electrostatic mechanism” is more favorable than the “charge relay mechanism” owing to the nature of the enzyme as a biopolymer.

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.

The energetics of proton transfers in the methanethiol-imidazole-formic acid complex were analyzed from a quantum chemical point of view in order to elucidate the nature of the enzymatic reaction of papain and the electronic structure in the active site. Methanethiol, imidazole and formic acid were used as models of the side chains of Cys 25, His 159 and Asp 158. The calculations were performed within the closed-shell-LCAO-ab initio MO method by using a 4-31G basis set. The hydrogen-bonding structure including the anion form of the methanethiol, the cation form of the imidazole and the anion form of the formic acid was the most stable of the various hydrogen-bonding structures. This result is in good agreement with experiments on the electronic structure in the active site of papain, and it supports the mechanism of the enzymatic reaction proposed by Angelides and Fink.

Rotational barrier for borane monoammoniate was studied at the Hartree-Fock level using several Gaussian basis sets. Rotational barrier to nonrigid rotation was estimated to be 1.85 kcal/mol using the 6–31G ☆ basis set. Furthermore, energy decomposition of the barrier was performed. For the rigid rotation, the origin of the rotational barrier was dominantly due to the exchange repulsion term. On the contrary, for the nonrigid rotation, the electrostatic term was the most important contributor, and the exchange repulsion played rather minimal role. The nonrigid rotation is also explained as the two-step process of the rigid rotation and the geometry relaxation.

A novel method of estimating Hansch's hydrophobic constant (log P) by making use of the solvent-accessible surface area (SA) with correction for the hydrophilic effect of any polar moiety (SH) is proposed. The correlation coefficient (r) between observed and estimated values of log P was 0.995 with 138 miscellaneous compounds. The method can reproduce the differences of log P among geometrical isomers. Such differences are not calculable by using other available methods. An application of SA and SH to the regression analysis of water-solubility data of 156 different organic liquids gave r=0.981. The proposed method may offer new insight into the physicochemical nature of hydrophobic phenomena.

Summary Antibody-dependent cellular cytotoxicity (ADCC) was measured using 51Cr-labelled ATL derived cell lines as the target, peripheral mononuclear cells (PMNCs) from disease-free persons as the effector cells and heat-inactivated serum from patients with ATL or the IgG purified from this. The release of 51Cr was not usually demonstrated in the HTLV-1 non-producing ATL derived cell line (MT-1), but was evident in the HTLV-1 producing ATL derived cell lines (KT 252 and IT 607). The 51Cr-labelled MT-1 cells after induction of HTLV-1 retrovirus by 5-iodo-2′-deoxyuridine (IdUr), showed a remarkable target sensitivity in the ADCC assay. On the other hand, the 51Cr-labelled MT-1 cells after culture with IdUr and ATL patient's serum, had no ADCC sensitivity, The fresh ATL cells immediately separated from ATL patient's blood did not express HTLV-1 virus in the cell and had no ADCC sensitivity as the target. Based on these findings, an antigenic modulation on the ATL cell surface by ATL patient's serum is considered to be the possible mode of escape of ATL cells from ADCC.

The function of the hydrogen bond between His 57 and Asp 102 in serine proteases was studied by ab initio molecular orbital calculations on a model of the enzyme. The calculated hydrogen bond distance was 2.73A, which is in good agreement with the distances obtained by X-ray diffraction analyses of trypsin. The stabilization energy is -30.0 kcal/mol. The electrostatic interaction term is the dominant contributor. The polarization energy and the charge transfer energy are also significant. The charge transfer energy is largely due to charge transfer from the aspartic acid to the histidine. The lone pair molecular orbital (LPMO) level of the nitrogen of His 57 increases substantially due to the electrostatic interaction between the two molecules. As a result, the charge transfer interaction between Ser 195 and the LPMO increases, and the increase of LPMO level should play a role in lowering the barrier height of proton transfer from Ser 195 to His 57 in trypsin. The populations of the LPMO upon complex formation are changed due to the polarization term. The populations of the outer shell of the nitrogen LPMO increase substantially, and hence the stabilization energy between His 57 and Ser 195 will increase with complex formation between His 57 and Asp 102. On the basis of optimized calculations of the complex, it is suggested that the hydrogen-bond structure in trypsin involves the anion of Asp 102, the neutral form of His 57 and the neutral form of Ser 195.

The charge state of the His 57-Asp 102 couple in β-trypsin in the acid pH range was studied using the ab initio molecular orbital method. The result was in agreement with the NMR experiments which do not support the charge relay hypothesis proposed by Blow et al. It is remarkable that the charge state of His 57-Asp 102 couple is greatly affected by the environment around the catalytic triad.

The active sites of serine proteases include the hydrogen bonds structure composed of serine, histidine, and aspartate. The two reaction mechanisms for the hydrogen bonds structure have been proposed. One is the charge relay system proposed by Blow et al. The other mechanism is the hydrogen bonding system proposed by Wang, and Polgar and Bender. In this system the form of Ser (anion)-His (cation)-Asp (anion) is stabilized with the proton transfer from Ser to His. In order to decide between two mechanisms, LCAO MO SCF ab initio calculations by using a 4-31G basis set were carried out. In the active site of the trypsin with pancreatic trypsin inhibitor (PTI), the potential energy surface of the two proton transfers from Ser to His and from His to Asp is described. The form of Ser (neutral)-His (neutral)-Asp (anion) is most stable. The form of Ser (anion)-His (cation)-Asp (anion) is much more stable than that of Ser (anion)-His (neutral)-Asp-(neutral). Accordingly, the hydrogen bonding system is supported as the catalytic mechanism of serine proteases.

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.

Methotrexate (MTX) is a high affinity inhibitor of the reaction catalyzed by dihydrofolate reductase (DHFR) from Lactobacillus casei. MTX is a folic acid analogue, being effective in the treatment of neoplastic disease in humans. In order to understand the mechanism of its inhibition from an electrostatic point of view, we calculated electrostatic potentials originating from MTX, NADPH (co-factor)-DHFR complex, NADPH and DHFR, individually. The mutual relationships between MTX and DHFR-NADPH and between NADPH and DHFR were then visualized and compared with the simultaneous geometrical analysis, using a raster computer graphic technique. Complete complementalities of the electrostatic potential and the field electrostatic potential are communicated between MTX and the NADPH-DHFR complex and between NADPH and DHFR.

Molecular orbital studies were carried out on α-chymotrypsin, papain, and thiolsubtilisin by using the complete neglect of differential overlap/2 method. By comparison between α-chymotrypsin and papain, the following results were obtained : (1) The proton transfer barrier from Cys-25 (neutral) to His-57 (neutral) is lower than that from Ser-195 (neutral) to His-57 (anion) in the"charge relay system"of α-chymotrypsin. (2) The active site of papain does not have the"charge relay system"and asparagine facilitates the proton transfer from cysteine to histidine. The results for thiolsubtilisin were as follows : (1) The hydrogen bond system structure in the active site is aspartate (neutral)-histidine (neutral)-cysteine (anion), and the"charge relay system"is broken by cysteine in place of serine. (2) Even though the effect of solvent is present, cysteine anion is stable. A"charge relay system"composed of aspartate, histidine, and water dimer was proposed from the calculations by using water dimer in place of serine.

A three-dimensional model of human renin has been constructed based on the assumption that the overall folding of the aspartyl proteases is very similar. As a reference, we used penicillopepsin, the structure of which has been reported at a resolution of 1.8 A, and its main chain was traced to build a model of renin. The resulting structure seems to be stable from the hydrophobic and hydrophilic viewpoints. Comparison of the tertiary structure of human renin with that of penicillopepsin and mouse renin suggests the existence of a high structural homology as well as differences in the molecular geometry of the active sites that may influence the substrate specificity. The asparagine side chains in the glycosidation signal of Asn-X-Thr are exposed on the surface. Moreover, the site in human renin that corresponds to the proteolytic cleavage site in mouse renin also appears to be exposed on the surface so as to be easily scissored during the maturation process. The insertions and deletions of amino acid residues were found to arise on the surface, and in some places they occurred in complementary manners. Models of molecular complexes between human renin and renin inhibitor were constructed to understand the interacting modes that indicate how new renin inhibitors develop. Inhibitor-binding sites were directly assigned based on the models of the inhibitor-enzyme complex.

Ribonuclease A is well known from X-ray diffraction analysis as an enzyme which changes the conformation, when the substrate binds with the enzyme. Therefore amino acids were rotated in order to simulate the coupling movements of the amino acid side chains, water molecule, and the substrate. From the study of the movements of amino acids and the quantum chemioal calculations, a charge relay structure composed of Asp121, His 119 and a water molecule was simulated in the active site of the ribonuclease A. And the water located in the very position where the hydroxyl group of the water molecule can attack phosphorus of the substrate in the second step of the hydrolysis. Moreover it was reported that Lys 7 as well as Lys 41 may participate in the enzymatic reaction.

Sephadex G-75 gel filtration of cytoplasmic protein with BSP in vitro yielded elution patterns characteristic of the protein sources as liver, kidney, and small intestinal mucosa of rat and rabbit. In the both species, binding of sulfobromophthalein (BSP) with Y protein was observed to be predominant in the liver, and weak or not recognized in the kidney and small intestinal mucosa, as was to be expected. On the other hand, the BSP elution with Z protein fraction was appreciable in the rabbit kidney and the rat small intestinal mucosa, and it appeared that the role of Z protein on the fate of the organic anion in the body was not so simple. Two unknown fractions, Y'and Y"protein, were recognized in the BSP elution pattern with cytoplasmic protein of the rat kidney, and Y'protein was recognized in that of the rabbit kidney and small intestinal mucosa.

The mechanism of the enzymatic reaction of lactate dehydrogenase was studied from the quantum chemical point of view. The charge relay system composed of aspartate, histidine and lactate of the substrate or pyruvate enol of the inhibitor was assumed. The substrate or the inhibitor played a role as a proton donor like serine in the "charge relay system"of α-chymotrypsin. Moreover arginine 171 for the assumed charge relay system was very significant in order to lower the potential barrier of the proton transfer from the substrate or the inhibitor to histidine 195. Last the significance of the HOMOLUMO interaction between the substrate or the inhibitor and nicotinamide in NAD+was shown.