Conformational energy calculations of enzyme-substrate complexes of lysozyme. I. Energy minimization of monosaccharide and oligosaccharide inhibitors and substrates of lysozyme

Conformational energy calculations were performed on monosaccharide and oligosaccharide inhibitors and substrates of lysozyme to examine the preferred conformations of these molecules. A grid-search method was used to locate all of the low-energy conformational regions for N-acetyl-β-D -glycosamine (NAG), and energy minimization was then carried out in each of these regions. Three stable positions for the N-acetyl group have ben located, in two of which the plane of the amide unit is normal to the mean plane of the pyranosyl ring. Nine local energy minima were located for the —CH₂OH group. The positions of the two vicinal cis —OH groups are determined predominantly by interactions with either the —CH₂OH or the N-acetyl group. The most stable conformations of β-N-acetylmuramic acid (NAM) were determined from the study of the low-energy conformations of NAG. In the two stable orientations for the D -lactic acid side chain, the O—C—C′ plane (C′ being the carbon atom of the terminal carboxyl group) was found to be normal to the mean plane of the pyranosyl ring. The low-energy positions for the COOH group of NAM are determined mainly by interactions with neighboring groups. The conformational preferences of the α-anomers of NAG and NAM were also explored. The calculated conformation of the N-acetyl group for α-NAG was quite close to that determined by X-ray analysis. Two of the three lowest energy conformations of α-NAM are similar to the corresponding conformations of the β-anomer. A third low-energy structure, which has a hydrogen bond from the NH of the N-acetyl group to the C?O of the lactic acid group, corresponds very closely to the X-ray structure of this molecule. The preferred conformations of the disaccharides NAG–NAG, NAM–NAG and NAG–NAM were also investigated. Two preferred orientations of the reducing pyranosyl ring relative to the nonreducing ring were found for all of these disaccharides, both of which are close to the extended conformation. In one of these conformations, a hydrogen bond can form between the OH group attached to C₃ of the reducing sugar and the ring oxygen of the preceding residue. Each conformation can be stabilized further by a hydrogen bond between the CH₂OH (donor) of residue i + 1 and the C?O of residue i (acceptor). The interactions that determine conformations for all oligosaccharides containing both NAG and NAM are shown to be exclusively intraresidue and nearest neighbor interactions, so that it is possible to predict all stable conformations of oligosaccharides containing NAG and NAM in any sequence.     

Structural differences of matrix metalloproteinases. Homology modeling and energy minimization of enzyme-substrate complexes

Matrix metalloproteinases are extracellular enzymes taking part in the remodeling of extracellular matrix. The structures of the catalytic domain of MMP1, MMP3, MMP7 and MMP8 are known, but structures of enzymes belonging to this family still remain to be determined. A general approach to the homology modeling of matrix metalloproteinases, exemplified by the modeling of MMP2, MMP9, MMP12 and MMP14 is described. The models were refined using an energy minimization procedure developed for matrix metalloproteinases. This procedure includes incorporation of parameters for zinc and calcium ions in the AMBER 4.1 force field, applying a non-bonded approach and a full ion charge representation. Energy minimization of the apoenzymes yielded structures with distorted active sites, while reliable three-dimensional structures of the enzymes containing a substrate in active site were obtained. The structural differences between the eight enzyme-substrate complexes were studied with particular emphasis on the active site, and possible sites for obtaining selectivity among the MMP's are discussed. Differences in the P1' pocket are well-documented and have been extensively exploited in inhibitor design. The present work indicates that selectivity could be further improved by considering the P2 pocket as well.     

Conformation of histidine model peptides. I. Conformational energy calculations for L-alanyl-L-histidine diketopiperazine

Semiempirical conformational energy calculations were carried out for the cyclic dipeptide L -alanyl-L -histidine diketopiperazine. The results indicate that electrostatic effects are probably significant in determining the conformation assumed by this molecule. When the imidazole group is in its uncharged state the most stable conformations of the molecule are those with the imidazole ring folded over the diketopiperazine ring (χ¹ = 60°). Upon protonation of the imidazole group the folded conformation may be destabilized relative to conformations characterized by χ¹ positions near 180°.     

The binding of monosaccharide inhibitors to hen egg-white lysozyme by proton magnetic resonance at 270 MHz and analysis by ring-current calculations.

Studies of the binding of the four sugars alpha- and beta-N-acetyl-D-glucosamine (GlcNAc) and its alpha- and beta-methyl glycosides to hen egg-white lysozyme (EC 3.2.1.17) by means of high-resolution 1H n.m.r. at 270 MHz are reported. The details of the binding analyses are described in an Appendix. The results show that the sugars bind independently to more than one site in lysozyme. The apparent fully bound chemical shifts to the inhibitor proton signals show that, although the major binding modes are generally similar for the four sugars, the binding of alpha GlcNAc is distinct from that of alpha MeGlcNAc and beta MeClcNAc. The binding of beta GlcNAc is intermediate in character between these two modes. The observed shift changes of the inhibitor signals are correlated with the crystal structures of lysozyme-inhibitor complexes by the use of Johnson-Bovey ring-current calculations. Together with consideration of the chemical-shift anisotropy of the GlcNAc amide group, these suggest that GlcNAc-binding sites in solution are in subsites C and E. The calculations show also that the indole rings of Trp-62 and Trp-63 rotate towards subsite C on the binding of GlcNAc, whereas Trp-108 moves away slightly. These findings indicate a difference between the solution and tetragonal crystal forms of lysozyme-GlcNAc and lysozymes-beta MeGlcNAc complexes. In the crystal structure, binding of acetamido monosaccharides is only observed in subsite C, and binding in subsite E is prevented by crystal packing.     

Conformational energy calculations on alginic acid I. Helix parameters and flexibility of the homopolymers

Conformational energy maps have been calculated for the 1-4-linked dimers of β-D -mannuronic acid and α-L -guluronic acid. Helix parameters have been calculated for poly(mannuronic acid) and for poly(guluronic acid), which are in reasonable agreement with data from x-ray fiber diffraction studies of these polysaccharides. The flexibility of the homopolymers was investigated by calculating the characteristic ratios, i.e., the ratio of the mean-square end-to-end lengths of the unperturbed chains to the product of the number of residues in the chains and the virtual bond lengths. The general conclusions are that both polymers are very stiff and extended, but that poly(mannuronic acid) is less extended than poly(guluronic acid).     

Conformational energy calculations on alginic acid. II. Conformational statistics of the copolymers

Conformational energy maps have been calculated for α-D -mannuronic acid (1–4) α-L -guluronic acid and for α-L -guluronic acid (1–4) β-D -mannuronic acid. These have been used, together with maps previously calculated for the homomonomeric dimers, to estimate the characteristic ratios and Kuhn lengths of the alternating copolymer and of a stochastic copolymer similar in composition to that extracted from L. digitata.The results show that the alternating copolymer is less extended than either homopolymer. Kuhn lengths calculated for the stochastic copolymer agree well with experimental results on high ionic strength solutions of alginate isolated from L digitata.     

Conformational aspects of beta-trypsin interaction with substrates and pancreatic trypsin inhibitor. I. Conformational properties of residues in the enzyme-active center and the structure of a non-valent enzyme-substrate complex

The theoretical conformational analysis of potential surfaces of Ser-195, His-57, Asp-102 and Gln-192 side chains in the active center of native beta-trypsin has been carried out. The above residues are shown to exist in low-energy conformational states in the free enzyme and in its nonbonded substrate complexes. Interrelations between the flexibility of the residues and their catalytical functions were revealed. Conformational aspects of interaction of trypsin with N-acetyl-L-lysine and N-acetyl-L-alanyl--L-alanyl--L-lysyl--L-alanyl methyl amide and with the Gly-12--Ile-19 BPTI fragment were analysed. The productive binding of the substrate at the nonbonded complex stage is shown to take place exclusively in the lowest energy conformation of the enzyme-substrate complex. Basing on theoretical and experimental evidence, the problems of primary and secondary specificity of trypsin, and potential properties of the native protein to form a productive nonbonded complex and to react at the subsequent stages of the catalytical act are discussed. Conformational changes in the active center and interactions with a substrate are shown to result from stabilizing enzyme-substrate interactions. Trypsin inhibition by BPTI molecule does not take place at the nonbonded complex stage.     

Biosynthesis of lipid-linked oligosaccharides. I. Preparation of lipid-linked oligosaccharide substrates.

In order to purify the glycosyltransferases involved in the assembly of lipid-linked oligosaccharides and to be able to study the acceptor substrate specificity of these enzymes, methods were developed to prepare and purify a variety of lipid-linked oligosaccharides, differing in the structure of the oligosaccharide moiety. Thus, Man9 (GlcNAc)2-pyrophosphoryl-dolichol was prepared by isolation and enzymatic synthesis using porcine pancreatic microsomes, while Glc3Man9(GlcNAc)2-PP-dolichol was isolated from Madin-Darby canine kidney cells. Treatment of these oligosaccharide lipids with a series of selected glycosidases led to the preparation of Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6(Man alpha 1,3)Man alpha 1,6]Man beta 1,4GlcNAc beta 1,4GlcNAc-PP-dolichol; Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6]Man beta 1,4GlcNAc beta 1, 4GlcNac-PP-dolichol; and Man alpha 1,6(Man alpha 1,3)Man alpha 1, 6[Man alpha 1,3]Man beta 1,4GlcNAc-beta 1,4GlcNAc-PP-dolichol. The preparation, isolation, and characterization of each of these lipid-linked oligosaccharide substrates are described.     

Conformational analysis of phosphatides: Mapping and minimization of the intramolecular energy

Empirical intramolecular energy calculations were carried out on molecular fragments related to phosphatides in order to find the preferred conformations. The energy was mapped as a function of several pairs of torsional angles in progressively larger molecular fragments, with energy minimization being carried out at each map point with respect to other significant variables. The energy mapping results were used as starting points for energy minimization on diheptanoyl L-α-phosphatidic acid-C, which consisted of the named molecule plus a carbon atom attached to one of the phosphate oxygens. It was found that there are 6 pairs of values for 2 of the torsional angles at the 3-way branch point in the glyceryl group which give sterically acceptable conformations; only 4 of these are compatible with lipid bilayer structure in that they can give a parallel arrangement of the acyl chains. The several acceptable conformations of the phosphate and acyl ester groups within each of these conformational classes are enumerated. The results obtained may be used as a guide for further experimental and theoretical work on phosphatide structures.     

Conformational characteristics of the RNA subunit ApA from energy minimization studies

In an attempt to better our understanding of the conformational stabilities in RNAs, an intensive theoraticl study has been carried out on one of its dimeric subunits, ApA, using an improved set of atom-atom interaction energy parameters and an improved version of energy-minimization technique. The C(3′)0endo and the C(2′)-endo sugar ApA units were sperately considered and 38 probable conformations have been analyzed in each case. The total potential energy, comprising nonbonded, electrostatic, and torsional contributions, was minimized by varying all seven relevant dihedral angles simumtaneously. The result reveal that 17 conformations in the case of C(3′)-endo sugar ApA and 7 confomations in the case of C(2′)-endo sugar ApA unit, the lowest energy conformation corresponds to a nonhelical structure and the A-RNA and the Watson-Crick-yype conformations lie at energy levels of about 0.5 and 1.0 Kcal/mo., respectively, above the lowest energy found. For ApA with the lops of different types in the backbone and they all differ in energies by about 3.5 Kcal/mol with refrence to the lowest energy founs. It is noted that the order ofmprefrence of the base stacking is observed in the A-RNA and the Watson-Crick type conformers. The ApA unit with C(2′)-endo sugar is forced to assume phosphodiester conformations with large deviations fom the expected staggered conformations compared to the ApA unit with C(3′)-endo sugar. The result obtained for ApA are discussed with refrence to those previously obtained for the dApdA unit. Te theoretical predictions are compared with the experimental data on the tRNA^Phe crystal, as well as those on fibrous RNAs and RNa subunitlike crystal structures. This study brings out many important aspects of the conformational stability of ApA which have been missed by studies made by others on this system.     

Conformational stability of ribonuclease A complexes with specific inhibitors

Isotermic unfolding of ribonuclease A, phosphopyridoxyl-8Lys41-RNAase A and complexes of the enzyme with cytidine, 2'-CMP, 3'-CMP, 3'-AMP and with the phosphoric ester of 1-(omega-oxypropyl)-cytosine in presence of urea has been studied. The stabilization of the protein structure resulting from the complex formation was shown to be determined by the ligand nucleobase binding. The comparison of the results obtained with those known from the literature suggests, that binding and catalytic zones of the enzyme active site form an integrated network system which is substained by multipoint contacts between the constituents. The change in the state of any part within the enzyme active state affects the energetics of the whole protein globule.     

Alternate substrates of dopamine beta-hydroxylase. I. Kinetic investigations of benzyl cyanides as substrates and inhibitors

A series of benzyl cyanide analogs have been studied as substrates and inhibitors of dopamine beta-hydroxylase to extend our initial report (Baldoni, J. M., and Villafranca, J. J. (1980) J. Biol. Chem. 255, 8987-8990) which showed that p-hydroxybenzyl cyanide was a suicide substrate of dopamine beta-hydroxylase. Thus, the appVmax values for benzyl cyanide analogs decrease in the order p-OH greater than m-OH greater than H much greater than p-OCH3,m-OCH3; the m-OH, m-OCH3 and p-OCH3 analogs are competitive inhibitors versus tyramine in initial velocity studies. The Vmax values for tyramine and p-hydroxybenzyl cyanide are nearly identical at saturating O2 and ascorbate (pH 5.0, 37 degrees C) but the Km for O2 is 0.14 and 2.8 mM, respectively, with tyramine and p-hydroxybenzyl cyanide. Studies of the pH dependence of log V/K for tyramine show two pKa values of 5.2 and 5.8 while for m-hydroxybenzyl cyanide the values are 5.3 and 5.9. The log Vmax profile shows one pKa of 5.9 with tyramine as substrate. Thus, nearly identical enzymic groups are involved in binding and/or catalysis with these two substrates. All the benzyl cyanide analogs are suicide inactivators of dopamine beta-hydroxylase. With m-hydroxybenzyl cyanide, the partition between catalysis and inactivation (kcat/kinact) changed from approximately 600 to approximately 17 as the pH varied from 5.0 to 6.7. The log kinact versus pH profile shows one pKa value of 6.0, suggesting that an enzymic group must be deprotonated for maximal inactivation. Copper was essential for the suicide inactivation of dopamine beta-hydroxylase by benzyl cyanides and kinetic studies of partially inhibited dopamine beta-hydroxylase (approximately 50%) showed that inactive enzyme molecules were completely inactive. The following papers in this series discuss the partial reactivation of suicide-inhibited dopamine beta-hydroxylase and the stoichiometry of inactivation by benzyl cyanide analogs.     

Statine and its derivatives. Conformational studies using nuclear magnetic resonance spectrometry and energy calculations

The conformations of derivatives of 3(S)-hydroxy-4(s)-amino-6-methylheptanoic acid (statine) and its analogs have been studied by n.m.r. in chloroform and in dimethyl sulfoxide, and by molecular mechanics calculations. The data obtained from these studies indicate that: 1) the coupling constant between NH and C4H is large, suggesting that the dihedral angle (theta) is near 165 degrees or 0 degree; 2) the coupling constant between C4H-C3H is small, indicating a vicinal bond angle of approximately 90 degrees; 3) the hydrogen deuterium exchange rate of statine amide protons is slow; however, the rate is dependent upon the electron withdrawing substituents adjacent to the amide NH's; 4) intramolecular hydrogen bonds involving the NH of the statine amide group do not stabilize conformations of single amino acid derivatives. Based on the n.m.r. results, four possible conformations of Boc-statine-OMe in solution are possible. MM1 calculations indicate one conformation is especially likely.