Molecular Modeling and docking of Wheat Hydroquinone Glucosyl transferase by using Hydroquinone,Phenyl phosphorodiamate and n-(n butyl) Phosphorothiocic Triamide as Inhibitors

In agriculture high urease activity during urea fertilization causes substantial environmental and economical problems by releasing abnormally large amount of ammonia into the atmosphere which leads to plant damage as well as ammonia toxicity. All over the world, urea is the most widely applied nitrogen fertilizer. Due to the action of enzyme urease; urea nitrogen is lost as volatile ammonia. For efficient use of nitrogen fertilizer, urease inhibitor along with the urea fertilizer is one of the best promising strategies. Urease inhibitors also provide an insight in understanding the mechanism of enzyme catalyzed reaction, the role of various amino acids in catalytic activity present at the active site of enzyme and the importance of nickel to this metallo enzyme. By keeping it in view, the present study was designed to dock three urease inhibitors namely Hydroquinone (HQ), Phenyl Phosphorodiamate (PPD) and N-(n-butyl) Phosphorothiocic triamide (NBPT) against Hydroquinone glucosyltransferase using molecular docking approach. The 3D structure of Hydroquinone glucosyltransferase was predicted using homology modeling approach and quality of the structure was assured using Ramachandran plot. This study revealed important interactions among the urease inhibitors and Hydroquinone glucosyltransferase. Thus, it can be inferred that these inhibitors may serve as future anti toxic constituent against plant toxins.     

A new proposal for urease mechanism based on the crystal structures of the native and inhibited enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels.

BACKGROUND: Urease catalyzes the hydrolysis of urea, the final step of organic nitrogen mineralization, using a bimetallic nickel centre. The role of the active site metal ions and amino acid residues has not been elucidated to date. Many pathologies are associated with the activity of ureolytic bacteria, and the efficiency of soil nitrogen fertilization with urea is severely decreased by urease activity. Therefore, the development of urease inhibitors would lead to a reduction of environmental pollution, to enhanced efficiency of nitrogen uptake by plants, and to improved therapeutic strategies for treatment of infections due to ureolytic bacteria. Structure-based design of urease inhibitors would require knowledge of the enzyme mechanism at the molecular level. RESULTS: The structures of native and inhibited urease from Bacillus pasteurii have been determined at a resolution of 2.0 A by synchrotron X-ray cryogenic crystallography. In the native enzyme, the coordination sphere of each of the two nickel ions is completed by a water molecule and a bridging hydroxide. A fourth water molecule completes a tetrahedral cluster of solvent molecules. The enzyme crystallized in the presence of phenylphosphorodiamidate contains the tetrahedral transition-state analogue diamidophosphoric acid, bound to the two nickel ions in an unprecedented mode. Comparison of the native and inhibited structures reveals two distinct conformations of the flap lining the active-site cavity. CONCLUSIONS: The mode of binding of the inhibitor, and a comparison between the native and inhibited urease structures, indicate a novel mechanism for enzymatic urea hydrolysis which reconciles the available structural and biochemical data.     

Evaluation of indole esters as inhibitors of p60(c-Src) receptor tyrosine kinase and investigation of the inhibition using receptor docking studies

Several indole esters were tested as inhibitors of tyrosine kinase p60(c-Src). Compound (4) was found fairly active against the enzyme with IC50 = 1.34 microM. DOCK methodology was used to asses our inhibitors for their inhibitory potency against tyrosine kinase. The docking results showed that compounds (4), (25) and (26) were bound to the active site of the enzyme Lys 295 of p60(c-Src) tyrosine kinase. Both activity and docking studies showed a parallel result, with compound (4) having a better interaction with the enzyme active site and also greater activity than the other compounds, indicating a potential role as new lead inhibitor.     

Methodology and problems of protein-ligand docking: case study of dihydroorotate dehydrogenase,thymidine kinase,and phosphodiesterase 4

The docking methodology was applied to three different therapeutically interesting enzymes: human dihydroorotate dehydrogenase (DHODH), Herpes simplex virus type I thymidine kinase (HSV1 TK) and human phosphodiesterase 4 (PDE4). Programs FlexX, AutoDock and DOCK where used. The three targets represent three distinct cases. For DHODH and HSV1 TK, the binding modes of substrate and inhibitors within the active site are known, while the binding orientation of cAMP within PDE4 has been solely hypothesized. Active site of DHODH is mainly hydrophobic and the binding mode of the inhibitor brequinar was used as a template for evaluating the docking strategies. The presence of cofactors revealed to be crucial for the definition of the docking site. The HSV1 TK active site is small and polar and contains crystal water molecules and ATP. Docking of thymidine and aciclovir (ACV) within the active site was analyzed by keeping or removing water molecules. It showed the crucial role of water in predicting the binding of pyrimidines and purines. The crystal structure of PDE4 contains magnesium and zinc cations as well as catalytic water molecule but no ligand. Several docking experiments of cAMP and rolipram were performed and the results showed clear-cut dependence between the ligand orientation and the presence of metals in the active site. All three cases show specific problems of the docking methodology, depending on the character of the active site.     

Evaluation of Indole Esters As Inhibitors of p60c-Src Receptor Tyrosine Kinase and Investigation of the Inhibition Using Receptor Docking Studies

Several indole esters were tested as inhibitors of tyrosine kinase p60c-Src. Compound (4) was found fairly active against the enzyme with IC50=1.34?μM. DOCK methodology was used to asses our inhibitors for their inhibitory potency against tyrosine kinase. The docking results showed that compounds (4), (25) and (26) were bound to the active site of the enzyme Lys 295 of p60c-Src tyrosine kinase. Both activity and docking studies showed a parallel result, with compound (4) having a better interaction with the enzyme active site and also greater activity than the other compounds, indicating a potential role as new lead inhibitor.     

Dimensional mapping of the active site of cholesterol esterase with alkylboronic acid inhibitors

The cholesterol esterase-catalyzed hydrolysis of the water-soluble substrate p-nitrophenyl butyrate occurs via an acylenzyme mechanism, and is competitively inhibited by boronic acid transition state analog inhibitors. Accordingly, we undertook to dimensionally map the enzyme's active site via synthesis and characterization of a series of n-alkyl boronic acid inhibitors. The most potent of these is n-hexaneboronic acid, with a Ki = 13 +/- 1 microM, since inhibitor potency declines for both longer and shorter boronic acids. No inhibition is observed for methaneboronic acid and n-octaneboronic acid inhibits poorly, with a Ki of 7 mM. These results indicate that the ability of the enzyme to form tight complexes with boron-containing transition state analog inhibitors is sensitive to alkyl chain length. The trend in inhibitor potency is discussed in terms of substrate specificity of and transition state stabilization by cholesterol esterase, and has important implications for the design of optimal reversible inhibitors of the enzyme.     

Jack bean urease: the effect of active-site binding inhibitors on the reactivity of enzyme thiol groups

In view of the complexity of the role of the active site flap cysteine in the urease catalysis, in this work we studied how the presence of typical active-site binding inhibitors of urease, phenylphosphorodiamidate (PPD), acetohydroxamic acid (AHA), boric acid and fluoride, affects the reactivity of enzyme thiol groups, the active site flap thiol in particular. For that the inhibitor-urease complexes were prepared with excess inhibitors and had their thiol groups titrated with DTNB. The effects observed were analyzed in terms of the structures of the inhibitor-urease complexes reported in the literature. We found that the effectiveness in preventing the active site cysteine from the modification by disulfides, varied among the inhibitors studied, even though they all bind to the active site. The variations were accounted for by different extents of geometrical distortion in the active site that the inhibitors introduced upon binding, leaving the flap either open in AHA-, boric acid- and fluoride-inhibited urease, like in the native enzyme or closed in PPD-inhibited urease. Among the inhibitors, only PPD was found to be able to thoroughly protect the flap cysteines from the further reaction with disulfides, this apparently resulting from the closed conformation of the flap. Accordingly, in practical terms PPD may be regarded as the most suitable inhibitor for active-site protection experiments in inhibition studies of urease.     

Ligand-protein interaction in biomembrane carriers. The induced transition fit of transport catalysis

Carrier-linked transport through biomembranes is treated under the view of catalysis. As in enzymes, substrate-protein interaction yields catalytic energy in overcoming the activation barrier. At variance with enzymes, catalytic energy is concentrated on structural changes of the carrier rather than on the substrate destabilization for facilitating the global protein rearrangements during transport. A transition state is invoked in which the binding site assumes the best fit to the substrate, whereas in the two ground (internal and external) states, the fit is poor. The maximum binding energy released in the transition state provides catalytic energy to enable the large carrier protein transformations associated with transport. This "induced transition fit" (ITF) of carrier catalysis provides a framework of rules, concerning specificity, unidirectional versus exchange type transport, directing inhibitors to the ground state instead of the transition state, and excluding simultaneous chemical and transport catalysis (vectorial group translocation). The possible role of external energy sources (ATP and Deltapsi) in supplementing the catalytic energy is elucidated. The analysis of the structure-function relationship based on new carrier structures may be challenged to account for the workings of the ITF.     

Kinetic and structural characterization of urease active site variants

Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze urea hydrolysis at >10(14)-fold the spontaneous rate. To better define the enzyme mechanism, we examined the kinetics and structures for a suite of site-directed variants involving four residues at the active site: His320, His219, Asp221, and Arg336. Compared to wild-type urease, the H320A, H320N, and H320Q variants exhibit similar approximately 10(-)(5)-fold deficiencies in rates, modest K(m) changes, and disorders in the peptide flap covering their active sites. The pH profiles for these mutant enzymes are anomalous with optima near 6 and shoulders that extend to pH 9. H219A urease exhibits 10(3)-fold increased K(m) over that of native enzyme, whereas the increase is less marked ( approximately 10(2)-fold) in the H219N and H219Q variants that retain hydrogen bonding capability. Structures for these variants show clearly resolved active site water molecules covered by well-ordered peptide flaps. Whereas the D221N variant is only moderately affected compared to wild-type enzyme, D221A urease possesses low activity ( approximately 10(-)(3) that of native enzyme), a small increase in K(m), and a pH 5 optimum. The crystal structure for D221A urease is reminiscent of the His320 variants. The R336Q enzyme has a approximately 10(-)(4)-fold decreased catalytic rate with near-normal pH dependence and an unaffected K(m). Phenylglyoxal inactivates the R336Q variant at over half the rate observed for native enzyme, demonstrating that modification of non-active-site arginines can eliminate activity, perhaps by affecting the peptide flap. Our data favor a mechanism in which His219 helps to polarize the substrate carbonyl group, a metal-bound terminal hydroxide or bridging oxo-dianion attacks urea to form a tetrahedral intermediate, and protonation occurs via the general acid His320 with Asp221 and Arg336 orienting and influencing the acidity of this residue. Furthermore, we conclude that the simple bell-shaped pH dependence of k(cat) and k(cat)/K(m) for the native enzyme masks a more complex underlying pH dependence involving at least four pK(a)s.     

Crystal structure of two quaternary complexes of dethiobiotin synthetase, enzyme-MgADP-AlF3-diaminopelargonic acid and enzyme-MgADP-dethiobiotin-phosphate; implications for catalysis.

The crystal structures of two complexes of dethiobiotin synthetase, enzyme-diaminopelargonic acid-MgADP-AlF3 and enzyme-dethiobiotin-MgADP-Pi, respectively, have been determined to 1.8 A resolution. In dethiobiotin synthetase, AlF3 together with carbamylated diaminopelargonic acid mimics the phosphorylated reaction intermediate rather than the transition state complex for phosphoryl transfer. Observed differences in the binding of substrate, diaminopelargonic acid, and the product, dethiobiotin, suggest considerable displacements of substrate atoms during the ring closure step of the catalytic reaction. In both complexes, two metal ions are observed at the active site, providing evidence for a two-metal mechanism for this enzyme.     

产碱杆菌Alcaligenes sp.KS-85来源肌酸酶活性中心的关键氨基酸功能研究

肌酸酶(Creatinase,EC 3.5.3.3)水解肌酸生成尿素和肌氨酸,是肌酐多酶级联检测中的关键酶.为进一步解析产碱杆菌来源肌酸酶的催化机理,利用蛋白质同源建模、分子对接、丙氨酸扫描技术分析了酶与底物的相互作用,并聚焦于酶活性中心4个功能未知的保守位点Phe64、Asp102、Phe252、Phe321,通过将...     

Methodology and Problems of Protein‐Ligand Docking: Case Study of Dihydroorotate Dehydrogenase,Thymidine Kinase,and Phosphodiesterase 4

Abstract

The docking methodology was applied to three different therapeutically interesting enzymes: human dihydroorotate dehydrogenase (DHODH), Herpes simplex virus type I thymidine kinase (HSV1 TK) and human phosphodiesterase 4 (PDE4). Programs FlexX, AutoDock and DOCK where used. The three targets represent three distinct cases. For DHODH and HSV1 TK, the binding modes of substrate and inhibitors within the active site are known, while the binding orientation of cAMP within PDE4 has been solely hypothesized. Active site of DHODH is mainly hydrophobic and the binding mode of the inhibitor brequinar was used as a template for evaluating the docking strategies. The presence of cofactors revealed to be crucial for the definition of the docking site. The HSV1 TK active site is small and polar and contains crystal water molecules and ATP. Docking of thymidine and aciclovir (ACV) within the active site was analyzed by keeping or removing water molecules. It showed the crucial role of water in predicting the binding of pyrimidines and purines. The crystal structure of PDE4 contains magnesium and zinc cations as well as catalytic water molecule but no ligand. Several docking experiments of cAMP and rolipram were performed and the results showed clear‐cut dependence between the ligand orientation and the presence of metals in the active site. All three cases show specific problems of the docking methodology, depending on the character of the active site.     

Five atomic resolution structures of endothiapepsin inhibitor complexes: implications for the aspartic proteinase mechanism

Endothiapepsin is derived from the fungus Endothia parasitica and is a member of the aspartic proteinase class of enzymes. This class of enzyme is comprised of two structurally similar lobes, each lobe contributing an aspartic acid residue to form a catalytic dyad that acts to cleave the substrate peptide bond. The three-dimensional structures of endothiapepsin bound to five transition state analogue inhibitors (H189, H256, CP-80,794, PD-129,541 and PD-130,328) have been solved at atomic resolution allowing full anisotropic modelling of each complex. The active sites of the five structures have been studied with a view to studying the catalytic mechanism of the aspartic proteinases by locating the active site protons by carboxyl bond length differences and electron density analysis. In the CP-80,794 structure there is excellent electron density for the hydrogen on the inhibitory statine hydroxyl group which forms a hydrogen bond with the inner oxygen of Asp32. The location of this proton has implications for the catalytic mechanism of the aspartic proteinases as it is consistent with the proposed mechanism in which Asp32 is the negatively charged aspartate. A number of short hydrogen bonds (approximately 2.6 A) with ESD values of around 0.01 A that may have a role in catalysis have been identified within the active site of each structure; the lengths of these bonds have been confirmed using NMR techniques. The possibility and implications of low barrier hydrogen bonds in the active site are considered.     

Enzymatic mechanism of creatine amidinohydrolase as deduced from crystal structures.

Crystal structures of the enzyme creatine amidinohydrolase (creatinase, EC 3.5.3.3) with two different inhibitors, the reaction product sarcosine and the substrate creatine, bound have been analyzed by X-ray diffraction methods. With the inhibitor carbamoyl sarcosine, two different crystal forms at different pH values have been determined. An enzymatic mechanism is proposed on the basis of the eight structures analyzed. The enzyme binds substrate and inhibitor in a distorted geometry where the urea resonance is broken. His232 is the general base and acid, and acts as a proton shuttle. It withdraws a proton from water 377 and donates it to the N(3) atom of the guanidinium group. OH- 377 adds to the C(1) atom of the guanidinium group to form a urea hydrate. Proton withdrawal by His232 leads to products. The reaction product sarcosine binds to the active site in a reverse orientation. The free enzyme was found to have a bicarbonate bound to the active site.     

Systematic optimization model and algorithm for binding sequence selection in computational enzyme design

A systematic optimization model for binding sequence selection in computational enzyme design was developed based on the transition state theory of enzyme catalysis and graph‐theoretical modeling. The saddle point on the free energy surface of the reaction system was represented by catalytic geometrical constraints, and the binding energy between the active site and transition state was minimized to reduce the activation energy barrier. The resulting hyperscale combinatorial optimization problem was tackled using a novel heuristic global optimization algorithm, which was inspired and tested by the protein core sequence selection problem. The sequence recapitulation tests on native active sites for two enzyme catalyzed hydrolytic reactions were applied to evaluate the predictive power of the design methodology. The results of the calculation show that most of the native binding sites can be successfully identified if the catalytic geometrical constraints and the structural motifs of the substrate are taken into account. Reliably predicting active site sequences may have significant implications for the creation of novel enzymes that are capable of catalyzing targeted chemical reactions.     

Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1. Homology guided site-directed mutagenesis.

CYP73 enzymes are highly conserved cytochromes P450 in plant species that catalyse the regiospecific 4-hydroxylation of cinnamic acid to form precursors of lignin and many other phenolic compounds. A CYP73A1 homology model based on P450 experimentally solved structures was used to identify active site residues likely to govern substrate binding and regio-specific catalysis. The functional significance of these residues was assessed using site-directed mutagenesis. Active site modelling predicted that N302 and I371 form a hydrogen bond and hydrophobic contacts with the anionic site or aromatic ring of the substrate. Modification of these residues led to a drastic decrease in substrate binding and metabolism without major perturbation of protein structure. Changes to residue K484, which is located too far in the active site model to form a direct contact with cinnamic acid in the oxidized enzyme, did not influence initial substrate binding. However, the K484M substitution led to a 50% loss in catalytic activity. K484 may affect positioning of the substrate in the reduced enzyme during the catalytic cycle, or product release. Catalytic analysis of the mutants with structural analogues of cinnamic acid, in particular indole-2-carboxylic acid that can be hydroxylated with different regioselectivities, supports the involvement of N302, I371 and K484 in substrate docking and orientation.     

Enzymes as a special class of therapeutic target: clinical drugs and modes of action

Enzymes catalyze multistep chemical reactions and achieve phenomenal rate accelerations by matching protein and substrate chemical groups in the transition state. Inhibitors that take advantage of these chemical interactions are among the most potent and effective drugs known. Recently, three new enzyme targets have been validated by FDA approval of new enzyme inhibitor drugs. These include mitogen-activated protein kinase, renin, and dipeptidyl peptidase IV. The drugs against these enzymes engage important enzyme functional groups, such as the active site serine in dipeptidyl peptidase IV. Clinical and pre-clinical discovery programs also demonstrate the same theme, as evidenced by pM and fM transition state inhibitors of purine nucleoside phosphorylase, methylthioadenosine phosphorylase, and 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase, and covalent substrate trapping in leu-tRNA synthetase. The catalytic chemistry of enzymes is the key to designing potent inhibitors and makes them a special class of drug target.     

Structure and catalysis of acylaminoacyl peptidase: closed and open subunits of a dimer oligopeptidase

Acylaminoacyl peptidase from Aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. The monomer subunit is composed of one hydrolase and one propeller domain. Previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. Here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. Using different substrates, we have determined the pH dependence of specificity rate constants, the rate-limiting step of catalysis, and the binding of substrates and inhibitors. The catalysis considerably depended both on the kind of mutation and on the nature of the substrate. The results were interpreted in terms of alterations in the position of the catalytic histidine side chain as demonstrated with crystal structure determination of the native and two mutant structures (D524N and D524A). Unexpectedly, in the homodimeric structures, only one subunit displayed the closed form of the enzyme. The other subunit exhibited an open gate to the catalytic site, thus revealing the structural basis that controls the oligopeptidase activity. The open form of the native enzyme displayed the catalytic triad in a distorted, inactive state. The mutations affected the closed, active form of the enzyme, disrupting its catalytic triad. We concluded that the two forms are at equilibrium and the substrates bind by the conformational selection mechanism.     

Reactivity of the essential thiol of Klebsiella aerogenes urease. Effect of pH and ligands on thiol modification

The kinetics of Klebsiella aerogenes urease inactivation by disulfide and alkylating agents was examined and found to follow pseudo-first-order kinetics. Reactivity of the essential thiol is affected by the presence of substrate and competitive inhibitors, consistent with a cysteine located proximal to the active site. In contrast to the results observed with other reagents, the rate of activity loss in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) saturated at high reagent concentrations, indicating that DTNB must first bind to urease before inactivation can occur. The pH dependence for the rate of urease inactivation by both disulfide and alkylating agents was consistent with an interaction between the thiol and a second ionizing group. The resulting macroscopic pKa values for the 2 residues are less than 5 and 12. Spectrophotometric studies at pH 7.75 demonstrated that 2,2'-dithiodipyridine (DTDP) modified 8.5 +/- 0.2 mol of thiol/mol of enzyme or 4.2 mol of thiol/mol of catalytic unit. With the slow tight binding competitive inhibitor phenyl-phosphorodiamidate (PPD) bound to urease, 1.1 +/- 0.1 mol of thiol/mol of catalytic unit were protected from modification. PPD-bound DTDP-modified urease could be reactivated by dialysis, consistent with the presence of one thiol per active site. Analogous studies at pH 6.1, using the competitive inhibitor phosphate, confirmed the presence of one protected thiol per catalytic unit. Under denaturing conditions, 25.5 +/- 0.3 mol of thiol/mol of enzyme (Mr = 211, 800) were modified by DTDP.     

X-ray structure of the R69D phosphatidylinositol-specific phospholipase C enzyme: insight into the role of calcium and surrounding amino acids in active site geometry and catalysis

Phosphatidylinositol-specific phospholipase Cs (PLCs) are a family of phosphodiesterases that catalyze the cleavage of the P-O bond via transesterification using the internal hydroxyl group of the substrate as a nucleophile, generating the five-membered cyclic inositol phosphate as an intermediate or product. To better understand the role of calcium in the catalytic mechanism of PLCs, we have determined the X-ray crystal structure of an engineered PLC enzyme from Bacillus thuringiensis to 2.1 A resolution. The active site of this enzyme has been altered by substituting the catalytic arginine with an aspartate at position 69 (R69D). This single-amino acid substitution converted a metal-independent, low-molecular weight enzyme into a metal ion-dependent bacterial PLC with an active site architecture similar to that of the larger metal ion-dependent mammalian PLC. The Ca(2+) ion shows a distorted square planar geometry in the active site that allows for efficient substrate binding and transition state stabilization during catalysis. Additional changes in the positions of the catalytic general acid/general base (GA/GB) were also observed, indicating the interrelation of the intricate hydrogen bonding network involved in stabilizing the active site amino acids. The functional information provided by this X-ray structure now allows for a better understanding of the catalytic mechanism, including stereochemical effects and substrate interactions, which facilitates better inhibitor design and sheds light on the possibilities of understanding how protein evolution might have occurred across this enzyme family.