Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the synthetic sweetener aspartame, was synthesized, using thermolysin immobilized onto Amberlite XAD-7, both in ethyl acetate and in tert-amyl alcohol. The initial rates for synthesis of Z-AspPheOMe in the organic solvents were predicted on the basis of a model proposed for an aqueous/organic biphasic reaction and compared with the experimentally observed substrate concentration dependencies. The experimental synthetic rates using the enzyme immobilized at a high enzyme concentration were lower than the calculated ones over a wide range of the substrate concentration. It was suggested as a reason for this discrepancy that the enzyme molecules form compact aggregates and those existing inside the aggregates cannot be utilized for reaction. The experimental results with the enzyme immobilized at a low concentration in ethyl acetate coincided well with the calculated ones. On the other hand, when tert-amyl alcohol was used, the experimental results were different in tendency irrespective of the amount of enzyme loaded, probably due to the fact that a distinct water phase does not exist around the enzyme aggregates inside the support.
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PMID:Kinetic analysis for synthesis of a dipeptide precursor using an immobilized enzyme in water-immiscible organic solvents. 1623 27

The metal-dependent deacetylase LpxC catalyzes the first committed step of lipid A biosynthesis in Gram-negative bacteria. Accordingly, LpxC is an attractive target for the development of inhibitors that may serve as potential new antibiotics for the treatment of Gram-negative bacterial infections. Here, we report the 2.7 A resolution X-ray crystal structure of LpxC complexed with the substrate analogue inhibitor TU-514 and the 2.0 A resolution structure of LpxC complexed with imidazole. The X-ray crystal structure of LpxC complexed with TU-514 allows for a detailed examination of the coordination geometry of the catalytic zinc ion and other enzyme-inhibitor interactions in the active site. The hydroxamate group of TU-514 forms a bidentate chelate complex with the zinc ion and makes hydrogen bond interactions with conserved active site residues E78, H265, and T191. The inhibitor C-4 hydroxyl group makes direct hydrogen bond interactions with E197 and H58. Finally, the C-3 myristate moiety of the inhibitor binds in the hydrophobic tunnel of the active site. These intermolecular interactions provide a foundation for understanding structural aspects of enzyme-substrate and enzyme-inhibitor affinity. Comparison of the TU-514 complex with cacodylate and imidazole complexes suggests a possible substrate diphosphate binding site and highlights residues that may stabilize the tetrahedral intermediate and its flanking transition states in catalysis. Evidence of a catalytic zinc ion in the native zinc enzyme coordinated by H79, H238, D242, and two water molecules with square pyramidal geometry is also presented. These results suggest that the native state of this metallohydrolase may contain a pentacoordinate zinc ion, which contrasts with the native states of archetypical zinc hydrolases such as thermolysin and carboxypeptidase A.
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PMID:Mechanistic inferences from the binding of ligands to LpxC, a metal-dependent deacetylase. 1680 Jun 20

Aminopeptidase N from Escherichia coli is a broad specificity zinc exopeptidase belonging to aminopeptidase clan MA, family M1. The structures of the ligand-free form and the enzyme-bestatin complex were determined at 1.5- and 1.6-A resolution, respectively. The enzyme is composed of four domains: an N-terminal beta-domain (Met(1)-Asp(193)), a catalytic domain (Phe(194)-Gly(444)), a middle beta-domain (Thr(445)-Trp(546)), and a C-terminal alpha-domain (Ser(547)-Ala(870)). The structure of the catalytic domain exhibits similarity to thermolysin, and a metal-binding motif (HEXXHX(18)E) is found in the domain. The zinc ion is coordinated by His(297), His(301), Glu(320), and a water molecule. The groove on the catalytic domain that contains the active site is covered by the C-terminal alpha-domain, and a large cavity is formed inside the protein. However, there exists a small hole at the center of the C-terminal alpha-domain. The N terminus of bestatin is recognized by Glu(121) and Glu(264), which are located in the N-terminal and catalytic domains, respectively. Glu(298) and Tyr(381), located near the zinc ion, are considered to be involved in peptide cleavage. A difference revealed between the ligand-free form and the enzyme-bestatin complex indicated that Met(260) functions as a cushion to accept substrates with different N-terminal residue sizes, resulting in the broad substrate specificity of this enzyme.
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PMID:Crystal structure of aminopeptidase N (proteobacteria alanyl aminopeptidase) from Escherichia coli and conformational change of methionine 260 involved in substrate recognition. 1688 66

Engineered extremely thermostable variants of the thermolysin-like protease from Bacillus stearothermophilus possessing an introduced disulfide bond G8C/N60C (double mutant, DM) and six additional amino acid substitutions in the exposed loop region 56-69 (Boilysin, BLN) have been probed with respect to stability toward water-miscible organic solvents and detergents. The solvent concentrations where 50% of enzyme activity were irreversibly lost (C(50)) decreased in the order methanol > 2-propanol > dimethylsulfoxide > dioxane > acetonitrile > dimethylformamide > acetone. The C(50) values were remarkably higher for the thermostable variants than for the wild-type enzymes. Therefore, the stabilization of this loop region also protects the molecule from irreversible inactivation by solvents, and inactivation seems to follow principally the same mechanism as thermal inactivation. However, in contrast to thermal inactivation where the corresponding T(50) values of DM and BLN differed by 10 K, the differences of the C(50) values of DM and BLN were not significant. Detergents had great effects on proteolytic activities which were dependent on the individual detergent and its concentration, but mostly without significant differences between the enzyme variants. These effects were inactivating (SDS, sulfobetaine) or strongly activating (CTAB, CHAPS). Triton X-100 and Tween 20 were activating or inactivating at low and high concentrations, respectively. In all detergents, stabilities of the enzymes were strongly decreased. However, the more thermostable variants were affected by the detergents to the same extent as the wild-type enzymes suggesting that the mechanism of detergent inactivation is different from that of thermal inactivation.
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PMID:The stability of engineered thermostable neutral proteases from Bacillus stearothermophilus in organic solvents and detergents. 1716 9

Prolonged heating of holo bovine alpha-lactalbumin (BLA) at 80 degrees C in pH 7 phosphate buffer in the absence of a thiol initiator improves the surface activity of the protein at the air:water interface, as determined by surface tension measurements. Samples after 30, 60, and 120 min of heating were analyzed on cooling to room temperature. Size-exclusion chromatography shows sample heterogeneity that increases with the length of heating. After 120 min of heating monomeric, dimeric, and oligomeric forms of BLA are present, with aggregates formed from disulfide bond linked hydrolyzed protein fragments. NMR characterization at pH 7 in the presence of Ca2+ of the monomer species isolated from the sample heated for 120 min showed that it consisted of a mixture of refolded native protein and partially folded protein and that the partially folded protein species had spectral characteristics similar to those of the pH 2 molten globule state of the protein. Circular dichroism spectroscopy showed that the non-native species had approximately 40% of the alpha-helical content of the native state, but lacked persistent tertiary interactions. Proteomic analysis using thermolysin digestion of three predominant non-native monomeric forms isolated by high-pressure liquid chromatography indicated the presence of disulfide shuffled isomers, containing the non-native 61-73 disulfide bond. These partially folded, disulfide shuffled species are largely responsible for the pronounced improvement in surface activity of the protein on heating.
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PMID:Heat treatment of bovine alpha-lactalbumin results in partially folded, disulfide bond shuffled states with enhanced surface activity. 1767 69

Enzymes, a class of highly efficient and specific catalysts in Nature, dictate a myriad of reactions that constitute various cascades in biological systems. Self-assembly, a process prevalent in Nature, also plays important roles in biology, from maintaining the integrity of cells to performing cellular functions and inducing abnormalities that cause disease. To explore enzyme-regulated molecular self-assembly in an aqueous medium will help to understand and control those important biological processes. On the other hand, certain small organic molecules self-assemble in water to form molecular nanofibers and result in a hydrogel, which is referred to as a "supramolecular hydrogel" (and the small molecules are referred to as "supramolecular hydrogelators"). Supramolecular hydrogelators share common features, such as amphiphilicity and supramolecular interactions (pi-pi interactions, hydrogen bonding, and charge interactions among the molecules, among others) that result in nanostructures and form the three-dimensional networks as the matrices of hydrogels. In this Account, we discuss the use of enzymes to trigger and control the self-assembly of small molecules for hydrogelation, which takes place in vitro or in vivo, extra- or intracellularly. Using phosphatase, thermolysin, beta-lactamase, and phosphatase/kinase as examples, we illustrate the design and application of enzyme-catalyzed or -regulated formation of supramolecular hydrogels that offer a new strategy for detecting the activity of enzymes, screening for enzyme inhibitors, typing bacteria, drug delivery systems, and controlling the fate of cells. Since the expression and distribution of enzymes differ by the types and states of cells, tissues, and organs, using an enzymatic reaction to convert precursors into hydrogelators that self-assemble into nanofibers as the matrices of the hydrogel, one can control the delivery, function, and response of a hydrogel according to a specific biological condition or environment, thus providing an accessible route to create sophisticated materials for biomedicine. Particularly, intracellular enzymatic hydrogelation of small molecules offers a unique means for scientists to integrate molecular self-assembly with inherent enzymatic reactions inside cells for developing new biomaterials and therapeutics at the supramolecular level and improving the basic understanding of dynamic molecular self-assembly in water.
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PMID:Enzymatic hydrogelation of small molecules. 1820 23

The spatial and temporal properties of water and ions in bionanoporous materials-protein crystals-have been investigated using molecular dynamics simulations. Three protein crystals are considered systematically with different morphologies and chemical topologies: tetragonal lysozyme, orthorhombic lysozyme, and tetragonal thermolysin. It is found that the thermal fluctuations of C(alpha) atoms in the secondary structures of protein molecules are relatively weak due to hydrogen bonding. The solvent-accessible surface area per residue is nearly identical in the three protein crystals; the hydrophobic and hydrophilic residues in each crystal possess approximately the same solvent-accessible surface area. Water distributes heterogeneously and has different local structures within the biological nanopores of the three protein crystals. The mobility of water and ions in the crystals is enhanced as the porosity increases and also by the fluctuations of protein atoms particularly in the two lysozyme crystals. Anisotropic diffusion is found preferentially along the pore axis, as experimentally observed. The anisotropy of the three crystals increases in the order: tetragonal thermolysin < tetragonal lysozyme < orthorhombic lysozyme.
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PMID:Molecular dynamics simulations for water and ions in protein crystals. 1831 54

Aminopeptidase N from Escherichia coli is a M1 class aminopeptidase with the active-site region related to that of thermolysin. The enzyme has unusual specificity, cleaving adjacent to the large, nonpolar amino acids Phe and Tyr but also cleaving next to the polar residues Lys and Arg. To try to understand the structural basis for this pattern of hydrolysis, the structure of the enzyme was determined in complex with the amino acids L-arginine, L-lysine, L-phenylalanine, L-tryptophan, and L-tyrosine. These amino acids all bind with their backbone atoms close to the active-site zinc ion and their side chain occupying the S1 subsite. This subsite is in the form of a cylinder, about 10 A in cross-section and 12 A in length. The bottom of the cylinder includes the zinc ion and a number of polar side chains that make multiple hydrogen-bonding and other interactions with the alpha-amino group and the alpha-carboxylate of the bound amino acid. The walls of the S1 cylinder are hydrophobic and accommodate the nonpolar or largely nonpolar side chains of Phe and Tyr. The top of the cylinder is polar in character and includes bound water molecules. The epsilon-amino group of the bound lysine side chain and the guanidinium group of arginine both make multiple hydrogen bonds to this part of the S1 site. At the same time, the hydrocarbon part of the lysine and arginine side chains is accommodated within the nonpolar walls of the S1 cylinder. This combination of hydrophobic and hydrophilic binding surfaces explains the ability of ePepN to cleave Lys, Arg, Phe, and Tyr. Another favored substrate has Ala at the P1 position. The short, nonpolar side chain of this residue can clearly be bound within the hydrophobic part of the S1 cylinder, but the reason for its facile hydrolysis remains uncertain.
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PMID:Structural basis for the unusual specificity of Escherichia coli aminopeptidase N. 1841 62

Cross-linked polyurethane (PU) was prepared for entrapping thermolysin. Using the immobilized thermolysin (IT), Z-L-aspartic acid (ZA) was reacted with -Lphenylalanine methyl ester (L-PM) in water-saturated ethyl acetate to give only alpha-Z-L-aspartylL-phenylalanine methyl ester (alpha-ZAPM). Ninety-four percent conversion of alpha-ZAPM was obtained for 30 h of reaction at 40 degrees C when 46 mg of enzyme was entrapped. PU support prepared from polypropylene glycol (#2000) showed better properties than from polypropylene (#1000) and polyethylene (#1000). Addition of polyol could increase the gel fraction of PU. The IT PU-ll-G-3, prepared from 1/2 mole ratio of PPG (#2000)/glycerin, gave the highest gel fraction and best swelling, and 89.0% of residual activity was obtained after four times of reuse (72 h). The stability of immobilized thermolysin was good; the activity loss resulting from degradatin and leak of enzyme in each time of reuse were found only about 2%. The kinetics of immobilized thermolysin-catalyzed condensation reaction of ZA with L-PM in water-saturated ethyl acetate was found to be first order in L-PM and the Lineweaver-Burk plot of 1/V against 1/[ZA] yields a straight line, showing that the reaction involves consecutive reactions of ZA and L-PM with the immobilized enzyme and with the ZA-immobilized enzyme complex, with the second reaction being the rate determining step.
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PMID:Synthesis of aspartame precursor: alpha-L-aspartyl-L-phenylalanine methyl ester in ethyl acetate using thermolysin entrapped in polyurethane. 1858 60

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the aspartame, and N-(benzyloxycarbonyl)-L-phenylalanyl-Lphenylalanine methyl ester (Z-PhePheOMe) were synthesized from the respective amino acid derivatives with an immobilized thermolysin (EC 3.4.24.4) in ethyl acetate. Various factors affecting the synthesis of these dipeptide precursors were clarified. The initial synthetic rate was the highest at the water content of 3.5% for both reactions. The substrate concentration dependencies of the initial synthetic rate of Z-AspkPheOMe and Z-PhePheOMe with the immobilized enzyme in ethyl acetate were different from those in an aqueous buffer solution saturated with ethyl acetate but similar to those in the aqueous/organic biphasic system using the free enzyme. Particularly, the initial synthetic rate of Z-AspPhOMe increased in order higher than first order with respect to the concentration of L-phenylalanine methyl ester (PheOMe), whereas it decreased sharply with the concentration of N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp). Such kinetic behavior could be explained by regarding the inside of the immobilized enzyme as being a biphasic mode composed from the organic phase and aqueous phase where the enzymatic reaction takes place. The reaction in the aqueous/organic biphasic system using the free enzyme could be simulated by taking into consideration the partition of the substrate and the initial rate of synthesis in the aqueous buffer saturated with ethyl acetate. Based on this analysis, the rate of reaction with the immobilized enzyme in ethyl acetate could also be predicted. Z-AsPheOMe and Z-PhePheOMe were synthesized by the fed-batch method where the acid component of the substrate was intermittently added during the course of reaction and by the batch method. In the synthesis of Z-AspPheOMe, the synthetic rate and maximum yield of reaction as well as the stability of the immobilized enzyme were higher in the fed-batch reaction than those in the batch reaction. In the synthesis of Z-PhePheOMe, the results obtained by both methods were similar. (c) 1994 John Wiley & Sons, Inc.
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PMID:Synthesis of dipeptide precursors with an immobilized thermolysin in ethyl acetate. 1861 23


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