Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Aminoacylase I from porcine kidney (EC 3.5.1.14) contains seven cysteine residues per subunit. Three sulfhydryl groups are accessible to modification by 4-hydroxymercuribenzoate (p-MB). The kinetics of the reaction suggest that only one of these groups affects acylase activity when modified by p-MB. Its reaction rate increases 2-3-fold when the essential metal ion of aminoacylase is removed. Modification of metal-free apoenzyme by N-ethylmaleimide (NEM) abolishes its activity without impairing Zn2+ binding. This indicates that the sulfhydryl group reacting with NEM is not directly coordinated to the metal. DTNB (5,5'-Dithio-bis(2-nitrobenzoate), Ellman's reagent) also modifies three sulfhydryl groups per subunit. In this case, the reactivities of native aminoacylase and apoenzyme are not significantly different. N-Hydroxy-2-aminobutyrate, a strong aminoacylase inhibitor, substantially increases the reactivity of the slowest reacting sulfhydryl in both native enzyme and metal-free aminoacylase. It appears that binding of the inhibitor or removal of the metal ion induces conformational changes of the amino-acylase active site that render a buried sulfhydryl group more accessible to modification.
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PMID:Reactivities of sulfhydryl groups in native and metal-free aminoacylase I. 277 87

To examine the effect of changing the amide bond of anandamide (5, AN) to a less hydrolyzable moiety, analogues 1a-1l, 2a-2c, 3a-3c, and 4a-4h were synthesized from commercially available arachidonyl alcohol or arachidonic acid and tested for their pharmacological activity. Arachidonyl ethers 1a-1k were obtained through the coupling of the arachidonyl mesylate (6) (generated from the mesylation of arachidonyl alcohol) with the appropriate alcohol in potassium hydroxide. Arachidonyl ether 1l was obtained through the phase-transfer coupling of arachidonyl alcohol with 2-(2-iodoethoxy)tetrahydropyran (which was generated from its bromide) followed by cleavage of the tetrahydropyran group with Dowex resin. Arachidonyl carbamates 2a-2c were obtained through the coupling of arachidonyl alcohol with the appropriate isocyanates. Norarachidonyl carbamates 3a-3c and ureas 4a-4h were obtained through the coupling of the norarachidonyl isocyanate (generated from arachidonic acid using diphenyl phosphorazidate and triethylamine upon heating) with the appropriate alcohols and amines, respectively. AN analogues 1-3 have shown poor binding affinities to the CB1 receptor and fail to produce significant pharmacological effect at doses up to 30 mg/kg. Several ether analogues 1 were also evaluated in the CB2 binding assay and were found to be of low affinity. However, norarachidonyl urea analogues 4 have shown generally good binding affinities to the CB1 receptor (Ki = 55-746 nM) and pharmacological activity with AN-like profiles. The most potent analogue of this series is the 2-fluoroethyl analogue 4f which binds 2 times better than AN and was more active in several mouse behavioral assays. It was also observed that urea analogues 4a and 4g, which have weak binding affinities to the CB1 receptor (Ki = 436 and 347 nM, respectively), produced surprisingly potent pharmacological activity. These urea analogues have also shown hydrolytic stability toward the amidase enzymes, responsible for the primary degradation pathway of anandamide, in binding affinity assays in the absence of the enzyme inhibitor PMSF.
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PMID:Unique analogues of anandamide: arachidonyl ethers and carbamates and norarachidonyl carbamates and ureas. 1035 5

An analysis of the X-ray structure of cilastatin bound to membrane dipeptidase, together with docking studies, is presented here to reveal how a simple amide may act as a high-affinity, reversible, amidase inhibitor. Cilastatin binds as a normal substrate and is orientated in a perfect near-attack conformer for formation of a tetrahedral intermediate with the zinc-bound water/hydroxide. This intermediate is fated, however, only to revert to its starting components as scission of the amide bond is prevented by the precise fit of cilastatin within the active site. The cilastatin alkyl end groups that are tightly buttressed against amino acid residues on opposite sides of the active site, are aligned along the C-N reaction coordinate axis thereby preventing collapse of the intermediate via rupture of the C-N bond. Such a feature could have more general applicability in the explicit design of substrate variants as selective, tight-binding, and reversible inhibitors.
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PMID:A substrate variant as a high-affinity, reversible inhibitor: insight from the X-ray structure of cilastatin bound to membrane dipeptidase. 1261 84

Isoaspartyl dipeptidase from Escherichia coli functions in protein degradation by catalyzing the hydrolysis of beta-L-isoaspartyl linkages in dipeptides. The best substrate for the enzyme reported thus far is iso-Asp-Leu. Here we report the X-ray analysis of the enzyme in its resting state and complexed with aspartate to 1.65 and 2.1 A resolution, respectively. The quaternary structure of the enzyme is octameric and can be aptly described as a tetramer of dimers. Each subunit folds into two distinct domains: the N-terminal region containing eight strands of mixed beta-sheet and the C-terminal motif that is dominated by a (beta,alpha)(8)-barrel. A binuclear zinc center is located in each subunit at the C-terminal end of the (beta,alpha)(8)-barrel. Ligands to the binuclear metal center include His 68, His 70, His 201, His 230, and Asp 285. The two zincs are bridged by a carboxylated lysine residue (Lys 162) and a solvent molecule, most likely a hydroxide ion. The product of the reaction, aspartate, binds to the enzyme by displacing the bridging solvent with its side chain functional group. From this investigation it is proposed that the reaction mechanism of the enzyme proceeds through a tetrahedral intermediate and that the bridging solvent attacks the re face of the carbonyl carbon of the scissile peptide bond. This structural analysis confirms the placement of isoaspartyl dipeptidase into the urease-related amidohydrolase superfamily.
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PMID:High-resolution X-ray structure of isoaspartyl dipeptidase from Escherichia coli. 1271 28

Direct hydroxide attack on the scissile carbonyl of the substrate has been suggested as a likely mechanism for esterase antibodies elicited by phosphonate haptens, which mimic the transition states for the alkaline hydrolysis of esters.1 The unique amidase activity of esterase antibody 43C9 has been attributed to nucleophilic attack by an active-site histidine residue.2 Yet, the active site of 43C9 is strikingly similar to those of other esterase antibodies, particularly 17E8. We have carried out quantum mechanical calculations, molecular dynamics simulations, and free energy calculations to assess the mechanism involving direct hydroxide attack for 43C9. Results support this mechanism and suggest that the mechanism is plausible for other antiphosphonate antibodies that catalyze the hydrolysis of (p-nitro)phenyl esters.
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PMID:Direct hydroxide attack is a plausible mechanism for amidase antibody 43C9. 1286 1

Isoaspartyl dipeptidase (IAD) is a member of the amidohydrolase superfamily and catalyzes the hydrolytic cleavage of beta-aspartyl dipeptides. Structural studies of the wild-type enzyme have demonstrated that the active site consists of a binuclear metal center positioned at the C-terminal end of a (beta/alpha)(8)-barrel domain. Steady-state kinetic parameters for the hydrolysis of beta-aspartyl dipeptides were obtained at pH 8.1. The pH-rate profiles for the hydrolysis of beta-Asp-Leu were obtained for the Zn/Zn-, Co/Co-, Ni/Ni-, and Cd/Cd-substituted forms of IAD. Bell-shaped profiles were observed for k(cat) and k(cat)/K(m) as a function of pH for all four metal-substituted forms. The pK(a) of the group that must be unprotonated for catalytic activity varied according to the specific metal ion bound in the active site, whereas the pK(a) of the group that must be protonated for catalytic activity was relatively independent of the specific metal ion present. The identity of the group that must be unprotonated for catalytic activity was consistent with the hydroxide that bridges the two divalent cations of the binuclear metal center. The identity of the group that must be protonated for activity was consistent with the free alpha-amino group of the dipeptide substrate. Kinetic constants were obtained for the mutant enzymes at conserved residues Glu77, Tyr137, Arg169, Arg233, Asp285, and Ser289. The catalytic properties of the wild-type and mutant enzymes, coupled with the X-ray crystal structure of the D285N mutant complexed with beta-Asp-His, are consistent with a chemical reaction mechanism for the hydrolysis of dipeptides that is initiated by the polarization of the amide bond via complexation to the beta-metal ion of the binuclear metal center. Nucleophilic attack by the bridging hydroxide is facilitated by abstraction of its proton by the side chain carboxylate of Asp285. Collapse of the tetrahedral intermediate and cleavage of the carbon-nitrogen bond occur with donation of a proton from the protonated form of Asp285.
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PMID:Mechanism of the reaction catalyzed by isoaspartyl dipeptidase from Escherichia coli. 1588 50

Isoaspartyl dipeptidase (IAD) is a binuclear metalloenzyme and a member of the amidohydrolase superfamily. This enzyme catalyzes the hydrolytic cleavage of beta-aspartyl dipeptides. The pH-rate profiles for the hydrolysis of beta-Asp-Leu indicates that catalysis is dependent on the ionization of two groups; one that ionizes at a pH approximately 6 and the other approximately 9. The group that must be ionized for catalysis is directly dependent on the identity of the metal ion bound to the active site. This result is consistent with the ionization of the hydroxide that bridges the two divalent cations. In addition to the residues that interact directly with the divalent cations there are two other residues that are highly conserved and found within the active site: Glu-77 and Tyr-137. Mutation of Tyr-137 to phenylalanine reduced the rate of catalysis by three orders of magnitude. The three dimensional X-ray structure of the Y137F mutant did not show any significant conformation changes relative to the three dimensional structure of the wild-type enzyme. The positioning of the side-chain phenolic group of Tyr-137 in the active site of IAD is consistent with the stabilization of the tetrahedral adduct concomitant with nucleophilic attack by the hydroxide that bridges the two divalent cations. Mutation of Glu-77 resulted in the reduction of catalytic activity by five orders of magnitude. The three dimensional structure of the E77Q mutant did not show any significant conformational changes in the mutant relative to the three dimensional structure of the wild-type enzyme. The positioning of the side-chain carboxylate of Glu-77 is consistent with the formation of an ion pair interaction with the free alpha-amino group of the substrate.
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PMID:Functional significance of Glu-77 and Tyr-137 within the active site of isoaspartyl dipeptidase. 1628 85

The tandem conversion process involving nitrile hydratase- and amidase-producing microorganisms has potential for use in the treatment of acetonitrile-containing wastes. In that process, the acetamide hydrolysis step catalyzed by amidase is very slow compared with the acetonitrile hydration step catalyzed by nitrile hydratase, and a small amount of acetamide remains in the resulting solution. This study aimed to improve the efficiency of the acetamide hydrolysis step. An amidase-producing microorganism, Rhodococcus sp. S13-4, was newly obtained, whose use enabled rapid acetamide degradation. Though residual acetamide was still detected, it was successfully reduced by the addition of cation/anion mixed ion exchange resin or calcium hydroxide after the acetamide hydrolysis reaction using Rhodococcus sp. S13-4 cells. This result implies that acetamide hydrolysis and acetamide formation are in equilibrium. The incubation of Rhodococcus sp. S13-4 cells with high concentrations of ammonium acetate produced acetamide. The purified amidase from Rhodococcus sp. S13-4 revealed the acetamide formation activity (specific activity of 30.6 U/mg protein). This suggests that the amidase-catalyzed amide formation may cause the remaining of acetamide in the acetonitrile conversion process.
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PMID:Remaining acetamide in acetonitrile degradation using nitrile hydratase- and amidase-producing microorganisms. 1713 68

NagA is a member of the amidohydrolase superfamily and catalyzes the deacetylation of N-acetyl-d-glucosamine-6-phosphate. The catalytic mechanism of this enzyme was addressed by the characterization of the catalytic properties of metal-substituted derivatives of NagA from Escherichia coli with a variety of substrate analogues. The reaction mechanism is of interest since NagA from bacterial sources is found with either one or two divalent metal ions in the active site. This observation indicates that there has been a divergence in the evolution of NagA and suggests that there are fundamental differences in the mechanistic details for substrate activation and hydrolysis. NagA from E. coli was inactivated by the removal of the zinc bound to the active site and the apoenzyme reactivated upon incubation with 1 equiv of Zn2+, Cd2+, Co2+, Mn2+, Ni2+, or Fe2+. In the proposed catalytic mechanism the reaction is initiated by the polarization of the carbonyl group of the substrate via a direct interaction with the divalent metal ion and His-143. The invariant aspartate (Asp-273) found at the end of beta-strand 8 in all members of the amidohydrolase superfamily abstracts a proton from the metal-bound water molecule (or hydroxide) to promote the hydrolytic attack on the carbonyl group of the substrate. A tetrahedral intermediate is formed and then collapses with cleavage of the C-N bond after proton transfer to the leaving group amine by Asp-273. The lack of a solvent isotope effect by D2O and the absence of any changes to the kinetic constants with increases in solvent viscosity indicate that net product formation is not limited to any significant extent by proton-transfer steps or the release of products. N-Trifluoroacetyl-d-glucosamine-6-phosphate is hydrolyzed by NagA 26-fold faster than the corresponding N-acetyl derivative. This result is consistent with the formation or collapse of the tetrahedral intermediate as the rate limiting step in the catalytic mechanism of NagA.
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PMID:N-Acetyl-D-glucosamine-6-phosphate deacetylase: substrate activation via a single divalent metal ion. 1756 47

Monofunctional histidinol phosphate phosphatase from Thermus thermophilus HB8, which catalyzes the dephosphorylation of l-histidinol phosphate, belongs to the PHP family, together with the PHP domain of bacterial DNA polymerase III and family X DNA polymerase. We have determined the structures of the complex with a sulfate ion, the complex with a phosphate ion, and the unliganded form at 1.6, 2.1, and 1.8 A resolution, respectively. The enzyme exists as a tetramer, and the subunit consists of a distorted (betaalpha)7 barrel with one linker and one C-terminal tail. Three metal sites located on the C-terminal side of the barrel are occupied by Fe1, Fe2, and Zn ions, respectively, forming a trinuclear metal center liganded by seven histidines, one aspartate, one glutamate, and one hydroxide with two Fe ions bridged by the hydroxide. In the complexes, the sulfate or phosphate ion is coordinated to three metal ions, resulting in octahedral, trigonal bipyramidal, and tetrahedral geometries around the Fe1, Fe2, and Zn ions, respectively. The ligand residues are derived from the four motifs that characterize the PHP family and from two motifs conserved in histidinol phosphate phosphatases. The (betaalpha)7 barrel and the metal cluster are closely related in nature and architecture to the (betaalpha)8 barrel and the mononuclear or dinuclear metal center in the amidohydrolase superfamily, respectively. The coordination behavior of the phosphate ion toward the metal center supports the mechanism in which the bridging hydroxide makes a direct attack on the substrate phosphate tridentately bound to the two Fe ions and Zn ion to hydrolyze the phosphoester bond.
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PMID:Crystal structure of monofunctional histidinol phosphate phosphatase from Thermus thermophilus HB8. 1792 34


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