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The primary photochemical reaction of the light, oxygen, and voltage (LOV) domain of the blue-light photosensor YtvA of Bacillus subtilis were investigated using high-level QM(DFT/MRCI)/MM methods. After blue-light excitation, the Sγ atom of the reactive cysteine forms a covalent bond with the C4a of the flavin mononucleotide (FMN) ring. Two conformations for the side chain of reactive cysteine with occupancies of 70% (conf A) and 30% (conf B) are observed in the X-ray crystallographic structures of the YtvA-LOV (Moglich, A.; Moffat, K. J. Mol. Biol. 2007, 373, 112−126). In conf A, the thiol group is directed toward the dimethylbenzene moiety of the FMN ring whereas it is placed directly above the N5 atom of the FMN ring in conf B. Starting from both conformations, the singlet and triplet excited pathways were evaluated. The singlet states excited from conf A decay nonradiatively to the triplet states by intersystem crossing (ISC). After the formation of a neutral biradical, the triplet states cross over ...

Molecular dynamics simulations were performed to investigate the stereoselective interaction between an enantiomeric amino acid substrate (N-acylated-l-phenylalanine-p-nitrophenyl ester) and an l-histidine-containing dipeptide catalyst (Nα-(N-benzyloxycarbonyl-l-leucyl)-l-histidine) in the bilayer of cationic surfactants (N,N-bisdodecyl-N,N-dimethylammonium chloride). We found that a catalyst–substrate complex, which had an interamide hydrogen bond, was formed spontaneously in vacuum at 500 K. This complex was found to be stable both in vacuum and in the bilayer membrane for 100 ps at 300 K. The distances between the hydrophobic side chains in the complex were consistent with experimental results. The interamide hydrogen bond was retained in the hydrophobic core of the membrane. These results suggest that the catalyst–substrate complex found in this work is relevant to the stereoselective hydrolysis of the l-enantiomer of the substrate.

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.

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.

Many investigations on the reaction mechanism of carboxypeptidase A (CPA) have shown that the zinc ion and Glu 270 in the active site are important in the catalytic reaction with a peptide substrate. X-Ray crystallographic studies of native CPA showed that the zinc ion is coordinated to His 69, Glu 72, His 196, and one water molecule and that the Zn-coordinated water molecule forms a hydrogen bond with Glu 270. The zinc-water-Glu 270 system of native CPA was analyzed by the ab initio SCF-LCAO-MO method. Some ligands of zinc are included in the MO calculations as point fractional charges. The results show that the Zn-coordinated water molecule acts as a proton donor to Glu 270, and that the electrostatic effect of Zn2+ and its ligands and the electron delocalization between Zn2+ and the water play a significant role in lowering the barrier height of proton transfer. We consider that the carbonyl group of the substrate, without breaking the hydrogen bond between Glu 270 and the Zn-coordinated water molecule, points towards a fifth coordination site slightly away from Zn2+, and that the water molecule itself is modified by its connection to Glu 270 in a way that favors the reaction.

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.

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.

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.

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 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.

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.

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.