Substrate-binding site of family 11 xylanase from Bacillus firmus K-1 by molecular docking.

The three-dimensional structure (3D structure) of Xyn11A, a family 11 xylanase from Bacillus firmus K-1, was obtained through homology modeling. To study the substrate-binding site of Xyn11A, six xylooligosaccharides, xylobiose to xyloheptaose (X2-X7), were docked into the active site of Xyn11A by molecular docking. Based on the docked energy and estimated free energy of binding combined with modeled enzyme-substrate complexes, the substrate-binding site of Xyn11A probably contained six subsites, defined as -3, -2, -1, +1, +2, and +3. Focus on possible stacking interaction presented seven aromatic residues, that played an important role in six subsites of Xyn11A such as Tyr165 (-3), Trp9 and Tyr69 (-2), Tyr80 (-1), Tyr65 (+1), Tyr88 (+2) and Tyr173 (+3). The bond-cleavage positions showed that X2 and X3 did not bind at the cleft (subsites -1 and +1) of Xyn11A. Related to the experiment, the end products of larchwood xylan hydrolysis by purified Xyn11A were X2 and X3. X2 and X3 acted as the end product inhibitors of Xyn11A.

A computational analysis of SARS cysteine proteinase-octapeptide substrate interaction: implication for structure and active site binding mechanism.

BACKGROUND: SARS coronavirus main proteinase (SARS CoVMpro) is an important enzyme for the replication of Severe Acute Respiratory Syndrome virus. The active site region of SARS CoVMpro is divided into 8 subsites. Understanding the binding mode of SARS CoVMpro with a specific substrate is useful and contributes to structural-based drug design. The purpose of this research is to investigate the binding mode between the SARS CoVMpro and two octapeptides, especially in the region of the S3 subsite, through a molecular docking and molecular dynamics (MD) simulation approach. RESULTS: The one turn alpha-helix chain (residues 47-54) of the SARS CoVMpro was directly involved in the induced-fit model of the enzyme-substrate complex. The S3 subsite of the enzyme had a negatively charged region due to the presence of Glu47. During MD simulations, Glu47 of the enzyme was shown to play a key role in electrostatic bonding with the P3Lys of the octapeptide. CONCLUSION: MD simulations were carried out on the SARS CoVMpro-octapeptide complex. The hypothesis proposed that Glu47 of SARS CoVMpro is an important residue in the S3 subsite and is involved in binding with P3Lys of the octapeptide.

Structure, dynamics and solvation of HIV-1 protease/saquinavir complex in aqueous solution and their contributions to drug resistance: molecular dynamic simulations.

As it is known that the understanding of the basic properties of the enzyme/inhibitor complex leads directly to enhancing the capability in drug designing and drug discovery. Molecular dynamics simulations have been performed to examine detailed information on the structure and dynamical properties of the HIV-1 PR complexed with saquinavir in the three protonated states, monoprotonates at Asp25 (Mono-25) and Asp25'(Mono-25') and diprotonate (Di-Pro) at both Asp25 and Asp25'. The obtained results support clinical data which reveal that Ile84 and Gly48 are two of the most frequent residues where mutation toward a protease inhibitor takes place. In contrast to the Ile84 mutation due to high displacement of Ile84 in the presence of saquinavir, source of the Gly48 mutation was observed to be due to the limited space in the HIV-1 PR pocket. The Gly48 was, on one side, found to form strong hydrogen bonds with saquinavir, while on the other side this residue was repelled by the hydrophobic Phe53 residue. In terms of inhibitor/enzyme binding, interactions between saquinavir and a catalytic triad of the HIV-1 PR were calculated using the ab initio method. The results show an order of the binding energy of Mono25

Improvement in hydrolytic antibody activity by change in haptenic structure from phosphate to phosphonate with retention of a common leaving-group determinant: evidence for the 'flexibility' hypothesis.

To investigate the hypothesis that decreased hapten flexibility may lead to increased catalytic antibody activity, we used two closely related immunogens differing only in the flexibility of the atomic framework around the structural motif of the haptens, analogous to the reaction centre of the corresponding substrates. Identical leaving-group determinants in the haptens and identical leaving groups in the substrates removed the ambiguity inherent in some data reported in the literature. Anti-phosphate and anti-phosphonate kinetically homogeneous polyclonal catalytic antibody preparations were compared by using carbonate and ester substrates respectively, each containing a 4-nitrophenolate leaving group. Synthetic routes to a new phosphonate hapten and new ester substrate were developed. The kinetic advantage of the more rigid anti-phosphonate/ester system was demonstrated at pH 8.0 by a 13-fold advantage in k(cat)/k(non-cat) and a 100-fold advantage in the proficiency constant, k(cat)/k (non-cat) x K(m). Despite these differences, the pH-dependences of the kinetic and binding characteristics and the results of chemical modification studies suggest closely similar catalytic mechanisms. The possible origin of the kinetic advantage of the more rigid hapten/substrate system is discussed.

Variation in the pH-dependent pre-steady-state and steady-state kinetic characteristics of cysteine-proteinase mechanism: evidence for electrostatic modulation of catalytic-site function by the neighbouring carboxylate anion.

The acylation and deacylation stages of the hydrolysis of N -acetyl-Phe-Gly methyl thionoester catalysed by papain and actinidin were investigated by stopped-flow spectral analysis. Differences in the forms of pH-dependence of the steady-state and pre-steady-state kinetic parameters support the hypothesis that, whereas for papain, in accord with the traditional view, the rate-determining step is the base-catalysed reaction of the acyl-enzyme intermediate with water, for actinidin it is a post-acylation conformational change required to permit release of the alcohol product and its replacement in the catalytic site by the key water molecule. Possible assignments of the kinetically influential p K (a) values, guided by the results of modelling, including electrostatic-potential calculations, and of the mechanistic roles of the ionizing groups, are discussed. It is concluded that Asp(161) is the source of a key electrostatic modulator (p K (a) 5.0+/-0.1) in actinidin, analogous to Asp(158) in papain, whose influence is not detected kinetically; it is always in the 'on' state because of its low p K (a) value (2.8+/-0.06).