Hydrolytic Water Research Articles

Molecular Orbital Study of the Interaction between MgATP and the Myosin Motor Domain: The Highest Occupied Molecular Orbitals Indicate the Reaction Site of ATP Hydrolysis

We constructed a model structure of Mg−adenosine triphosphate (MgATP) and the surrounding portion of myosin by using the X-ray crystal structure of the myosin motor domain bound to the stable MgATP analogue, MgADP−BeFx, and by adding hydrogen atoms and replacing beryllium with phosphorus and fluorine with oxygen. The positions of the hydrogen atoms were determined by optimization using the semiempirical molecular orbital program MOPAC 97. Analysis of the electronic states of the model structure revealed that the highest occupied molecular orbitals (HOMO) indicate that the reaction site of the ATP hydrolysis is the nonbridging γ-phosphoryl oxygen atoms and confirmed that H2O(1181) is the hydrolytic water. Thus, hypothetical mechanisms of the initial phase of the hydrolysis are proposed.

Characterization of metal-substituted Klebsiella aerogenes urease.

Urease possesses a dinuclear Ni active site with the protein providing a bridging carbamylated lysine residue as well as an aspartyl and four histidyl ligands. The apoprotein can be activated in vitro by incubation with bicarbonate/CO2 and Ni(II); however, only approximately 15% forms active enzyme (Ni-CO2-ureaseA), with the remainder forming inactive carbamylated Ni-containing protein (Ni-CO2-ureaseB). In the absence of CO2, apoprotein plus Ni(II) forms a distinct inactive Ni-containing species (Ni-urease). The studies described here were carried out to better define the metal-binding sites for the inactive Ni-urease and Ni-CO2-ureaseB species, and to examine the properties of various forms of Co-, Mn-, and Cu-substituted ureases. Xray absorption spectroscopy (XAS) indicated that the two Ni atoms present in the Ni-urease metallocenter are coordinated by an average of two histidines and 3-4 N/O ligands, consistent with binding to the usual enzyme ligands with the lysine carbamate replaced by solvent. Neither XAS nor electronic spectroscopy provided evidence for thiolate ligation in the inactive Ni-containing species. By contrast, comparative studies of Co-CO2-urease and its C319A variant by electronic spectroscopy were consistent with a portion of the two Co being coordinated by Cys319. Whereas the inactive Co-CO2-urease possesses a single histidyl ligand per metal, the species formed using C319A apoprotein more nearly resembles the native metallocenter and exhibits low levels of activity. Activity is also associated with one of two species of Mn-CO2-urease. A crystal structure of the inactive Mn-CO2-urease species shows a metallocenter very similar in structure to that of native urease, but with a disordering of the Asp360 ligand and movement in the Mn-coordinated solvent molecules. Cu(II) was bound to many sites on the protein in addition to the usual metallocenter, but most of the adventitious metal was removed by treatment with EDTA. Cu-treated urease was irreversibly inactivated, even in the C319A variant, and was not further characterized. Metal speciation between Ni, Co, and Mn most affected the higher of two pKa values for urease activity, consistent with this pKa being associated with the metal-bound hydrolytic water molecule. Our results highlight the importance of precisely positioned protein ligands and solvent structure for urease activity.

Relocation of the catalytic carboxylate group in class A beta-lactamase: the structure and function of the mutant enzyme Glu166-->Gln:Asn170-->Asp.

The hydrolysis of beta-lactam antibiotics by the serine-beta-lactamases proceeds via an acyl-enzyme intermediate. In the class A enzymes, a key catalytic residue, Glu166, activates a water molecule for nucleophilic attack on the acyl-enzyme intermediate. The active site architecture raises the possibility that the location of the catalytic carboxylate group may be shifted while still maintaining close proximity to the hydrolytic water molecule. A double mutant of the Staphylococcus aureus PC1 beta-lactamase, E166Q:N170D, was produced, with the carboxylate group shifted to position 170 of the polypeptide chain. A mutant protein, E166Q, without a carboxylate group and with abolished deacylation, was produced as a control. The kinetics of the two mutant proteins have been analyzed and the crystal structure of the double mutant protein has been determined. The kinetic data confirmed that deacylation was restored in E166Q:N170D beta-lactamase, albeit not to the level of the wild-type enzyme. In addition, the kinetics of the double mutant enzyme follows progressive inactivation, characterized by initial fast rates and final slower rates. The addition of ammonium sulfate increases the size of the initial burst, consistent with stabilization of the active form of the enzyme by salt. The crystal structure reveals that the overall fold of the E166Q:N170D enzyme is similar to that of native beta-lactamase. However, high crystallographic temperature factors are associated with the ohm-loop region and some of the side chains, including Asp170, are partially or completely disordered. The structure provides a rationale for the progressive inactivation of the Asp170-containing mutant, suggesting that the flexible ohm-loop may be readily perturbed by the substrate such that Asp170's carboxylate group is not always poised to facilitate hydrolysis.

Modulation of the Affinity and Selectivity of RGS Protein Interaction with Gα Subunits by a Conserved Asparagine/Serine Residue

The crystal structure of the complex between a G protein alpha subunit (Gi alpha 1) and its GTPase-activating protein (RGS4) demonstrated that RGS4 acts predominantly by stabilization of the transition state for GTP hydrolysis [Tesmer, J. J., et al. (1997) Cell 89, 251]. However, attention was called to a conserved Asn residue (Asn128) that could play a catalytic role by interacting, directly or indirectly, with the hydrolytic water molecule. We have analyzed the effects of several disparate substitutions for Asn128 on the GAP activity of RGS4 toward four G alpha substrates (Go, Gi, Gq, and Gz) using two assay formats. The results substantiate the importance of this residue but indicate that it is largely involved in substrate binding and that its function may vary with different G alpha targets. Various mutations decreased the apparent affinity of RGS4 for substrate G alpha proteins by several orders of magnitude, but had variable and modest effects on maximal rates of GTP hydrolysis when tested with different G alpha subunits. One mutation, N128F, that differentially decreased the GAP activity toward G alpha i compared with that toward G alpha q could be partially suppressed by mutation of the nearby residue in G alpha i to that found in G alpha q (K180P). Detection of GAP activities of the mutants was enhanced in sensitivity up to 100-fold by assay at steady state in proteoliposomes that contain heterotrimeric G protein and receptor.

Nuances of Mechanisms and Their Implications for Evolution of the Versatile β-Lactamase Activity: From Biosynthetic Enzymes to Drug Resistance Factors

β-Lactamases of classes A and C are believed to have evolved from bacterial enzymes involved in biosynthesis of the peptidoglycan, the so-called penicillin-binding proteins. All these enzymes undergo acylation at an active-site serine by β-lactam antibiotics as a common feature. However, the fate of the acyl-enzyme species is different for β-lactamases and penicillin-binding proteins; deacylation is rapid for the former, whereas it is slow for the latter. It is believed that the acquisition of the ability to deacylate the acyl-enzyme intermediate led to the evolution of β-lactamase activity, which is indispensable for the survival of bacteria in the face of challenge by β-lactam antibiotics. The mechanisms of deacylation of acyl-enzyme intermediates for β-lactamases are examined as a means to investigate structural factors in evolutionary descendency of classes A and C of β-lactamases from penicillin-binding proteins. It is known that in class A β-lactamases the hydrolytic water approaches the acyl-enzyme...

Structure of RGS4 Bound to AlF 4−-Activated G iα1: Stabilization of the Transition State for GTP Hydrolysis

RGS proteins are GTPase activators for heterotrimeric G proteins. We report here the 2.8 Å resolution crystal structure of the RGS protein RGS4 complexed with G iα1–Mg 2+–GDP–AlF 4 −. Only the core domain of RGS4 is visible in the crystal. The core domain binds to the three switch regions of G iα1, but does not contribute catalytic residues that directly interact with either GDP or AlF 4 −. Therefore, RGS4 appears to catalyze rapid hydrolysis of GTP primarily by stabilizing the switch regions of G iα1, although the conserved Asn-128 from RGS4 could also play a catalytic role by interacting with the hydrolytic water molecule or the side chain of Gln-204. The binding site for RGS4 on G iα1 is also consistent with the activity of RGS proteins as antagonists of G α effectors.

A point mutation leads to altered product specificity in beta-lactamase catalysis.

beta-Lactamases are the primary cause of beta-lactam antibiotic resistance in many pathogenic organisms. The beta-lactamase catalytic mechanism has been shown to involve a covalent acyl-enzyme. Examination of the structure of the class A beta-lactamase from Bacillus licheniformis suggested that replacement of Asn-170 by leucine would disrupt the deacylation reaction by displacing the hydrolytic water molecule. When N170L beta-lactamase was reacted with penicillins, a novel product was formed. We postulate that with leucine at position 170 the acyl-enzyme undergoes deacylation by an intramolecular rearrangement (rather than hydrolysis) to form a thiazolidine-oxazolinone as the initial product. The oxazolinone subsequently undergoes rapid breakdown leading to the formation of N-phenylacetylglycine and N-formylpenicillamine. This appears to be the first reported case where a point mutation leads to a change in enzyme mechanism resulting in a substantially altered product, effectively changing the product specificity of beta-lactamase into that of D-Ala-D-Ala-carboxypeptidase interacting with benzylpenicillin.

Quantification of the extent of attenuation of the rate of turnover chemistry of the TEM-1 β-lactamase by the α-1 R-hydroxyethyl group in substrates

The 6 α-1 R-hydroxyethyl group of imipenem, a clinically used carbapenem antibacterial agent, is believed to displace the hydrolytic water from its optimal position in the active site of class A β-lactamases. This interaction renders the molecule a poor substrate for these bacterial enzymes, hence preserving the antibacterial property of the antibiotic. The extent of the contribution of the 6 α-1 R-hydroxyethyl group in substrates toward stabilization of the antibiotic to the hydrolytic action of class A TEM-1 β-lactamase was studied by the synthesis and evaluation of two penicillanic acid derivatives, 6 α-(1 R-hydroxyethyl)penicillanic acid ( 2) and 6 β-(1 R-hydroxyethyl)penicillanic acid ( 3). The kinetic evaluation of the enzymic hydrolysis of these two penicillanic acid derivatives indicated that the 6 α-1 R-hydroxyethyl group imparts as much as 10 4-fold to the hydrolytic stability of the β-lactam substrate. The role of the 6α-1 R-hydroxythermal group in imparting hydrolytic stability to β-lactam antibiotics towards class A β-lactamase was investigated.

Evidence for Structural Elasticity of Class A β-Lactamases in the Course of Catalytic Turnover of the Novel Cephalosporin Cefepime

The mechanism of hydrolysis of cefepime, a novel cephalosporin, by the class A TEMpUC19 β-lactamase has been investigated. Models for the active-site binding of this antibiotic indicate severe steric interactions between the active site of the enzyme and the C7β function of cefepime. Specific interactions with the side-chain functions of Pro-167 and Asn-170, amino acids present in the Ω-loop spanning residues 164−179, have been singled out as important in the interactions with the antibiotic. These interactions displace the hydrolytic water (Wat-712) from its preferred position for the deacylation step. These observations are consistent with experimental evidence that deacylation is the rate-limiting step in the turnover of cefepime by this β-lactamase. Furthermore, it has been shown in circular-dichroic measurements that hydrolysis of cefepime by this β-lactamase is accompanied by an unprecedented relaxation of the structure of the enzyme in order to accommodate the bulky C7β side chain of the antibiotic...

Crystal Structure of 6α-(Hydroxymethyl)penicillanate Complexed to the TEM-1 β-Lactamase from Escherichia coli: Evidence on the Mechanism of Action of a Novel Inhibitor Designed by a Computer-Aided Process

The crystal structure of the complex of the TEM-1 β-lactamase from Escherichia coli inhibited by 6α-(hydroxymethyl)penicillanic acid (1) is reported herein. This is the first structure for an acyl-enzyme intermediate with a substrate reported for a native class A β-lactamase. This compound was designed and synthesized as a molecule that would acylate the active site of the enzyme, but would resist deacylation by virtue of the fact that its C6α hydroxymethyl moiety was expected to occupy the space near the hydrolytic water molecule (J. Am. Chem. Soc. 1995, 117, 11055). The crystal structure of the acyl-enzyme species is closely similar to one of the two energy-minimized acyl-enzyme models generated in the course of the design aspect of the work. The crystal structure provides evidence for a number of mechanistic features for the inhibition process and the ultimate recovery of the activity. Our results reported herein are consistent with the side-chain carboxylate of Glu-166 being the active-site basic func...

Elimination of the hydrolytic water molecule in a class A beta-lactamase mutant: crystal structure and kinetics.

Two site-directed mutant enzymes of the class A beta-lactamase from Staphylococcus aureus PC1 were produced with the goal of blocking the site that in the native enzyme is occupied by the proposed hydrolytic water molecule. The crystal structures of these two mutant enzymes, N170Q and N170M, have been determined and refined at 2.2 and 2.0 A, respectively. They reveal that the side chain of Gln 170 displaces the water molecule, whereas that of Met170 does not. In both cases, the catalytic rates with benzylpenicillin are reduced by 10(4) compared with the native enzyme. With nitrocefin, the N170Q mutant enzyme exhibits an approximately 800-fold reduced rate compared with the native enzyme and in addition, a fast initial burst with stoichiometry of 1 mol of degraded nitrocefin/mol of enzyme. Stopped-flow kinetic experiments establish that the rate constant of the burst is 250 s-1, a value comparable with the rate of acylation of the native enzyme. Two structurally based mechanisms that explain the kinetic properties of the N170Q beta-lactamase are proposed, both invoking a deacylation-impaired enzyme due to the elimination of the hydrolytic water molecule. The catalytic rate of the N170M mutant enzyme with nitrocefin is reduced by approximately 50-fold compared with the native enzyme, and the slow progressive inhibition that is revealed indicates that the hydrolysis proceeds via a branched pathway mechanism. This is consistent with the structural data that show that the water site is preserved and that Met170 occupies part of the space that is required for substrate binding. The short contacts between the substrate and the enzyme may lead to structure perturbation and inactivation.

The Refined Crystallographic Structure of a DD-Peptidase Penicillin-target Enzyme at 1.6 Å Resolution

The D-alanyl- D-alanine peptidase from Streptomycessp. R61 is a 37,500 dalton exocellular enzyme that has served as a model for membrane-bound peptidases that are involved in bacterial cell wall biosynthesis. Inhibition of these enzymes by β-lactam antibiotics ultimately leads to bacterial cell death. The X-ray crystal structure of the R61 D-alanyl- D-alanine peptidase has been solved using multiple isomorphous replacement, simulated annealing and least squares refinement. The space group and unit cell parameters are P2 12 12 1with a=51.1 Å, b= 67.3 Å and c=102.4 Å. The structure has been refined using 2σ data to 1.6 Å resolution with a crystallographic R-factor of 0.148. The model contains 347 residues (2938 atoms) and 254 solvent molecules. The overall temperature factor is 9.6 Å 2, and the estimated coordinate error is 0.14 Å. The protein consists of a single polypeptide chain organized into two regions. One region contains a nine-stranded antiparallel β-sheet with helices on both faces; this region includes both the amino and carboxyl termini. The second region is all helical. Sixty percent of the residues occur in helices or β-sheet. The reactive Ser62 is found between the two regions of the enzyme at the amino end of the protein's longest helix which begins with one turn of 3 10helix and continues with four turns of α-helix. The active site is an elongated pocket that contains four basic and four aromatic residues. An oxyanion hole is formed by Ser62 NH and Thr301 NH. The pocket also contains the few key residues that are conserved in all penicillin-binding proteins and β-lactamases. Two of these residues, Lys65 and Tyr159, are among the 16 side-chains that take on multiple conformations in the R61 crystal structure. Three of the 12 proline rings adopt two conformations which we believe has not been previously reported. There is no anionic acid equivalent to the catalytic Glu166 found in Class A β-lactamases. Two ordered water molecules (O507 and O644) are found buried in the active site and hydrogen-bonded to each other (2.6 Å). O507 could potentially act as the hydrolytic water molecule for deacylation.

3-Dimensional interactions between inositol monophosphatase and its substrates, inhibitors and metal ion cofactors

Experiment has shown that bovine inositol monophosphatase requires the presence of two Mg2+ cations for activity, the second of which interacts either directly or indirectly with peripheral O-atoms on the substrate. Here we describe the 3-D structure of the active enzyme di-magnesium ion complex and the modelled binding modes for five known and dissimilar substrates, D-inositol 1-phosphate, L-inositol 1-phosphate, inositol 4-phosphate, 2′-adenosine monophosphate and ethane-1,2-diol 1-phosphate The expected location and functional role of the second metal ion within the protein in the presence of each of these substrates is very similar. The predicted binding modes and interactions with the metal ions and the protein are also presented for a range of known inhibitors. These structures highlight the small differences that exist between substrates and inhibitors in the mode of coordination for the first Mg2+ ion within the enzyme and the large differences that exist in the mode of binding for the second Mg2 + ion. This analysis indicates that the hydrolytic water molecule is located on the second Mg2+ ion and that hydrolysis proceeds via a non-inline, direct displacement. The so called ‘catalytic’ oxygen functionality of substrates serves as a hydrogen-bond acceptor for the hydrolytic water molecule and holds it on the second Mg2+ ion, but does not appear to bind to the metal ion directly. Some of the predictions of this refined new mechanism have been tested by experiment and the binding conformations of new potential inhibitors which may be able to block the site for the nucleophile on the second Mg2 + ion have been examined. A comparison of the proposed pseudorotation mechanism with an alternative in-line displacement mechanism is presented.

Structures of active conformations of Gi alpha 1 and the mechanism of GTP hydrolysis

Mechanisms of guanosine triphosphate (GTP) hydrolysis by members of the G protein alpha subunit-p21ras superfamily of guanosine triphosphatases have been studied extensively but have not been well understood. High-resolution x-ray structures of the GTP gamma S and GDP.AlF4- complexes formed by the G protein Gi alpha 1 demonstrate specific roles in transition-state stabilization for two highly conserved residues. Glutamine204 (Gln61 in p21ras) stabilizes and orients the hydrolytic water in the trigonal-bipyramidal transition state. Arginine 178 stabilizes the negative charge at the equatorial oxygen atoms of the pentacoordinate phosphate intermediate. Conserved only in the G alpha family, this residue may account for the higher hydrolytic rate of G alpha proteins relative to those of the p21ras family members. The fold of Gi alpha 1 differs from that of the homologous Gt alpha subunit in the conformation of a helix-loop sequence located in the alpha-helical domain that is characteristic of these proteins; this site may participate in effector binding. The amino-terminal 33 residues are disordered in GTP gamma S-Gi alpha 1, suggesting a mechanism that may promote release of the beta gamma subunit complex when the alpha subunit is activated by GTP.

A Crystallographic Re-investigation into the Structure of Streptomyces griseus Proteinase A Reveals an Acyl-enzyme Intermediate

A molecular dynamics (MD) simulation on the contents of two asymmetric units of the crystal structure of the bacterial serine proteinase, Streptomyces griseus proteinase A (SGPA), resulted in four dihydrogen phosphate anions migrating to form a cluster in a solvent region far removed from the protein surface. In an effort to provide experimental verification for this unexpected phenomenon, native SGPA crystals were soaked in 2·0 M KH 2AsO 4; intensity data were collected and difference electron density maps examined for evidence of H 2AsO - 4 ion clusters. These maps did not corroborate the cluster observed in the MD simulation. They did, however, show positive electron density features located in the active site cavity that could be interpreted as a tetrapeptide. Gly-Ala-Ser (β-OH)Asp, covalently bonded to O γ of Ser195 as an acyl-enzyme intermediate. There was also a peak of electron density corresponding to an ideal position for the hydrolytic water in the deacylation reaction. This density is centred 3·1 Å from His57 N 2 and 2·7 Å from the carbonyl-carbon atom of the planar acyl-ester bond of the complex. The carbonyl-oxygen atom of the ester is located in the oxyanion pocket and participates in hydrogen bonds with Gly193 NH (2·6 Å) and Ser195 NH (3·0 Å).

The ability of time-resolved Laue crystallography to discriminate small changes in structure: appearance of a hydrolytic water molecule in trypsin catalysis

The ability of time-resolved Laue crystallography to discriminate small changes in structure: appearance of a hydrolytic water molecule in trypsin catalysis

Refined X-ray Structures of Haloalkane Dehalogenase at pH 6·2 and pH 8·2 and Implications for the Reaction Mechanism

The crystal structure of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 has been refined at 1·9 Å resolution at two different pH values, the pH of crystallization (pH 6·2) and the pH of optimal activity (pH 8·2), to final R-factors of 16·8% and 16·4%, respectively. Both models show good stereochemical quality. Two non-glycine residues have main-chain torsion angles that are located outside the "allowed" regions in a Ramachandran plot. One of them is the nucleophilic residue Asp124, which, together with the two other active site residues His289 and Asp260, is situated in an internal, predominantly hydrophobic cavity. The other residue, AsnI48, helps stabilize the conformations of two of these active-site residues, Asp124 and Asp260. Comparison of the models at pH 6·2 and pH 8·2 revealed one major structural difference. At pH6·2, a salt-bridge is present between the Nε2 atom of His289 and the Oδ1 atom of Asp124, while at pH 8·2, this salt-bridge is absent, indicating that the Nε2 atom of the histidine residue is mostly deprotonated at the pH of optimum activity. This is in agreement with the putative reaction mechanism in which the Oδ1 atom of Asp124 performs a nucleophilic attack on the substrate, resulting in an intermediate ester. This ester is subsequently cleaved by a hydrolytic water molecule. The high-resolution data sets clearly show the exact position of this water molecule. It is in an ideal position for donating a proton to the Nε2 atom of His289 and subsequently cleaving the covalently bound intermediate ester, releasing the alcohol product.Detailed investigation of both refined models showed a number of unusual structural features. Four out of 11 helices contain an internal proline residue other than in the first turn. Two other α-helices have adopted in their central part a 310 conformation. A novel four-residue turn between a helix and a strand, the αβ4 turn, is located at the site of the bend in the central eight-stranded β-sheet of the dehalogenase structure.

New vinyl ester monomers for emulsion polymers

New vinyl ester monomers are now available for the preparation of emulsion copolymers with improved hydrolytic stability and water resistance. These improved properties are the result of uniform copolymerization of hydrophobic monomers having approximately the same reactivity as vinyl acetate. Latex coatings with excellent gloss, water spot resistance, blocking and other desirable performance properties can now be obtained with vinyl acetate copolymers.

The hydrolytic water molecule in trypsin, revealed by time-resolved Laue crystallography.

Crystals of bovine trypsin were acylated at the reactive residue, serine 195, to form the transiently stable p-guanidinobenzoate. Hydrolysis of this species was triggered in the crystals by a jump in pH. The hydrolysis was monitored by three-dimensional Laue crystallography, resulting in three x-ray diffraction structures, all from the same crystal and each representing approximately 5 seconds of x-ray exposure. The structures were analyzed at a nominal resolution of 1.8 angstroms and were of sufficient quality to reproduce subtle features in the electron-density maps for each of the structures. Comparison of the structures before and after the pH jump reveals that a water molecule has positioned itself to attack the acyl group in the initial step of the hydrolysis of this transient intermediate.

A catalytically-impaired class A β-lactamase: 2 Å crystal structure and kinetics of the Bacillus licheniformis E166A mutant

In the beta-lactamase (penicillinase) of Bacillus licheniformis 749/C, site specific mutation of Glu166 to Ala caused a million-fold reduction of catalytic activity towards both penicillin and cephalosporin substrates and resulted in the stoichiometric accumulation of the acyl enzyme. The rate of deacylation generally slowed by as much as 10(-7) compared to the wild type. The acyl enzyme intermediate was observed by HPLC, but not by X-ray diffraction. The mutant was crystallized from methoxyPEG 5000 at pH 6.2 in space group P2(1) with Z = 4. Molecular replacement based on the wild type structure followed by refinement produced an R factor of 17.2% for 25,800 3 sigma data from 10 to 2 A. Deviations from bond and angle ideals are 0.005 A and 1.5 degrees respectively. The mutant differs very little from the wild type structure, with only 0.25 A (r.m.s.) differences in backbone atoms; the CD spectra and thermal stabilities of the two enzymes are identical. Changes in the positions of the reactive Ser70 and conserved Lys73 are not significant, suggesting that the proposed salt linkage to Glu166 in the wild type enzyme is weak or non-existent. The calculated solvent exposure of Ser70 and Lys73 increases slightly and a buried water molecule is now positioned near Lys73. The hydrolytic water seen in the native active site shifts markedly by 1.6 A, but is held in the active site by Asn170, which possibly becomes an ineffective substitute for Glu166 in activating the water for deacylation.