Evaluation at atomic resolution of the role of strain in destabilizing the temperature‐sensitive T4 lysozyme mutant Arg 96 → His

Mutant R96H is a classic temperature‐sensitive mutant of bacteriophage T4 lysozyme. It was in fact the first variant of the protein to be characterized structurally. Subsequently, it has been studied extensively by a variety of experimental and computational techniques, but the reasons for the loss of stability of the mutant protein remain controversial. In the crystallographic refinement of the mutant structure at 1.9 Å resolution one of the bond angles at the site of substitution appeared to be distorted by about 11^°, and it was suggested that this steric strain was one of the major factors in destabilizing the mutant. Different computationally‐derived models of the mutant structure, however, did not show such distortion. To determine the geometry at the site of mutation more reliably, we have extended the resolution of the data and refined the wildtype (WT) and mutant structures to be better than 1.1 Å resolution. The high‐resolution refinement of the structure of R96H does not support the bond angle distortion seen in the 1.9 Å structure determination. At the same time, it does confirm other manifestations of strain seen previously including an unusual rotameric state for His96 with distorted hydrogen bonding. The rotamer strain has been estimated as about 0.8 kcal/mol, which is about 25% of the overall reduction in stability of the mutant. Because of concern that contacts from a neighboring molecule in the crystal might influence the geometry at the site of mutation we also constructed and analyzed supplemental mutant structures in which this crystal contact was eliminated. High‐resolution refinement shows that the crystal contacts have essentially no effect on the conformation of Arg96 in WT or on His96 in the R96H mutant.     

Room temperature crystal structure of the fast switching M159T mutant of the fluorescent protein dronpa

The fluorescent protein Dronpa undergoes reversible photoswitching reactions between the bright “on” and dark “off” states via photoisomerization and proton transfer reactions. We report the room temperature crystal structure of the fast switching Met159Thr mutant of Dronpa at 2.0‐Å resolution in the bright on state. Structural differences with the wild type include shifted backbone positions of strand β8 containing Thr159 as well as an altered A‐C dimer interface involving strands β7, β8, β10, and β11. The Met159Thr mutation increases the cavity volume for the p‐hydroxybenzylidene‐imidazolinone chromophore as a result of both the side chain difference and the backbone positional differences. Proteins 2015; 83:397–402. © 2014 Wiley Periodicals, Inc.     

Crystal structure of the cataract‐causing P23T γD‐crystallin mutant

Up to now, efforts to crystallize the cataract‐associated P23T mutant of human γD‐crystallin have not been successful. Therefore, insights into the light scattering mechanism of this mutant have been exclusively obtained from solution work. Here we present the first crystal structure of the P23T mutant at 2.5 Å resolution. The protein exhibits essentially the same overall structure as seen for the wild‐type protein. Based on our structural data, we confirm that no major conformational changes are caused by the mutation, and that solution phase properties of the mutant appear exclusively associated with cataract formation. Proteins 2013. © 2013 Wiley Periodicals, Inc.     

Neutron crystallographic studies of T4 lysozyme at cryogenic temperature

Bacteriophage T4 lysozyme (T4L) has been used as a paradigm for seminal biophysical studies on protein structure, dynamics, and stability. Approximately 700 mutants of this protein and their respective complexes have been characterized by X‐ray crystallography; however, despite the high resolution diffraction limits attained in several studies, no hydrogen atoms were reported being visualized in the electron density maps. To address this, a 2.2 Å‐resolution neutron data set was collected at 80 K from a crystal of perdeuterated T4L pseudo‐wild type. We describe a near complete atomic structure of T4L, which includes the positions of 1737 hydrogen atoms determined by neutron crystallography. The cryogenic neutron model reveals explicit detail of the hydrogen bonding interactions in the protein, in addition to the protonation states of several important residues.     

Studying effects of different protonation states of His11 and His102 in ribose-5-phosphate isomerase of Trypanosoma cruzi: an example of cooperative behavior

Abstract

The Trypanosoma cruzi ribose-5-phosphate isomerase B (TcRpiB) is a crucial piece in the pentose phosphate pathway and thus is a potential drug target for treatment of Chagas’ disease. TcRpiB residues, such as Cys69, Asp45, Glu149 and Pro47, have confirmed their roles in substrate recognition, catalytic reaction and binding site conformation. However, the joint performance of His11 and His102, in the D-ribose-5-phosphate (R5P) in the catalysis is not well understood. In this work, we probed the influence of different protonation states of His11 and His102 on the behavior of the ligand R5P using molecular dynamics simulations, network analysis and thermodynamic integration. Simulations revealed that a protonated His11 combined with a neutral His102 (His11⁺?His102) was able to stabilize the ligand R5P in the binding site. Moreover, calculated relative free energy differences showed that when protonated His11 was coupled to a neutral His102 an exergonic process takes place. On the other hand, neutral His11 combined with a protonated His102 (His11?His102⁺), sampled conformations that resembled the catalyzed product D-ribulose-5-phosphate (Ru5P). Network analysis also demonstrated some peculiarities for these systems with some negatively correlated nodes in the binding site for His11?His102⁺, and exclusive suboptimal paths for His11⁺?His102. Therefore, the combined approach presented in this paper proposes two suitable protonation states for the TcRpiB catalytic mechanism, where an extra proton in either histidines might favor R5P binding or influence isomerization reaction to Ru5P. Our results may guide further in silico drug discovery studies.

Communicated by Ramaswamy H. Sarma     

Hydrogen-bond network and pH sensitivity in transthyretin: Neutron crystal structure of human transthyretin

Transthyretin (TTR) is a tetrameric protein associated with human amyloidosis. In vitro, the formation of amyloid fibrils by TTR is known to be promoted by low pH. Here we show the neutron structure of TTR, focusing on the hydrogen bonds, protonation states and pH sensitivities. A large crystal was prepared at pD 7.4 for neutron protein crystallography. Neutron diffraction studies were conducted using the IBARAKI Biological Crystal Diffractometer with the time-of-flight method. The neutron structure solved at 2.0 Å resolution revealed the protonation states of His88 and the detailed hydrogen-bond network depending on the protonation states of His88. This hydrogen-bond network is composed of Thr75, Trp79, His88, Ser112, Pro113, Thr118-B and four water molecules, and is involved in both monomer–monomer and dimer–dimer interactions, suggesting that the double protonation of His88 by acidification breaks the hydrogen-bond network and causes the destabilization of the TTR tetramer. In addition, the comparison with X-ray structure at pH 4.0 indicated that the protonation occurred to Asp74, His88 and Glu89 at pH 4.0. Our neutron model provides insights into the molecular stability of TTR related to the hydrogen-bond network, the pH sensitivity and the CH···O weak hydrogen bond.     

Second-site revertants of an inactive T4 lysozyme mutant restore activity by restructuring the active site cleft

Substitution of Thr26 by Gln in the lysozyme of bacteriophage T4 produces an enzyme with greatly reduced activity but essentially unaltered stability relative to wild type. Spontaneous second-site revertants of the mutant were selected genetically; two of them were chosen for structural and biochemical characterization. One revertant bears (in addition to the primary mutation) the substitution Tyr18----His, the other, Tyr18----Asp. The primary mutant and both revertant lysozyme genes were reconstructed in a plasmid-based expression system, and the proteins were produced and purified. The two revertant lysozymes exhibit enzymatic activities intermediate between wild type and the primary mutant; both also exhibit melting temperatures approximately 3 degrees C lower than either the wild type or the primary mutant. Crystals suitable for X-ray diffraction analysis were obtained from both revertant lysozymes, but not the primary mutant. Structures of the double mutant lysozymes were refined at 1.8-A resolution to crystallographic residuals of 15.1% (Tyr18----His) and 15.2% (Tyr18----Asp). Model building suggests that the side chain of Gln26 in the primary mutant is forced to protrude into the active site cleft, resulting in low catalytic activity. In contrast, the crystal structures of the revertants reveal that the double substitutions (Gln26 and His18, or Gln26 and Asp18) fit into the same space that is occupied by Thr26 and Tyr18 in the wild-type enzyme; the effect is a restructuring of the surface of the active site cleft, with essentially no perturbation of the polypeptide backbone. This restructuring is effected by a novel series of hydrogen bonds and electrostatic interactions that apparently stabilize the revertant structures.     

Atomic resolution crystallography of a complex of triosephosphate isomerase with a reaction‐intermediate analog: New insight in the proton transfer reaction mechanism

Enzymes achieve their catalytic proficiency by precisely positioning the substrate and catalytic residues with respect to each other. Atomic resolution crystallography is an excellent tool to study the important details of these geometric active‐site features. Here, we have investigated the reaction mechanism of triosephosphate isomerase (TIM) using atomic resolution crystallographic studies at 0.82‐Å resolution of leishmanial TIM complexed with the well‐studied reaction‐intermediate analog phosphoglycolohydroxamate (PGH). Remaining unresolved aspects of the reaction mechanism of TIM such as the protonation state of the first reaction intermediate and the properties of the hydrogen‐bonding interactions in the active site are being addressed. The hydroxamate moiety of PGH interacts via unusually short hydrogen bonds of its N1? O1 moiety with the carboxylate group of the catalytic glutamate (Glu167), for example, the distance of N1(PGH)‐OE2(Glu167) is 2.69 ± 0.01 Å and the distance of O1(PGH)‐OE1(Glu167) is 2.60 ± 0.01 Å. Structural comparisons show that the side chain of the catalytic base (Glu167) can move during the reaction cycle in a small cavity, located above the hydroxamate plane. The structure analysis suggests that the hydroxamate moiety of PGH is negatively charged. Therefore, the bound PGH mimics the negatively charged enediolate intermediate, which is formed immediately after the initial proton abstraction from DHAP by the catalytic glutamate. The new findings are discussed in the context of the current knowledge of the TIM reaction mechanism. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Crystal structures of the T4 phage beta-glucosyltransferase and the D100A mutant in complex with UDP-glucose: glucose binding and identification of the catalytic base for a direct displacement mechanism

T4 phage beta-glucosyltransferase (BGT) is an inverting glycosyltransferase (GT) that transfers glucose from uridine diphospho-glucose (UDP-glucose) to an acceptor modified DNA. BGT belongs to the GT-B structural superfamily, represented, so far, by five different inverting or retaining GT families. Here, we report three high-resolution X-ray structures of BGT and a point mutant solved in the presence of UDP-glucose. The two co-crystal structures of the D100A mutant show that, unlike the wild-type enzyme, this mutation prevents glucose hydrolysis. This strongly indicates that Asp100 is the catalytic base. We obtained the wild-type BGT-UDP-glucose complex by soaking substrate-free BGT crystals. Comparison with a previous structure of BGT solved in the presence of the donor product UDP and an acceptor analogue provides the first model of an inverting GT-B enzyme in which both the donor and acceptor substrates are bound to the active site. The structural analyses support the in-line displacement reaction mechanism previously proposed, locate residues involved in donor substrate specificity and identify the catalytic base.     

Structural and functional consequences of binding site mutations in transferrin: crystal structures of the Asp63Glu and Arg124Ala mutants of the N-lobe of human transferrin

Human transferrin is a serum protein whose function is to bind Fe(3+) with very high affinity and transport it to cells, for delivery by receptor-mediated endocytosis. Structurally, the transferrin molecule is folded into two globular lobes, representing its N-terminal and C-terminal halves, with each lobe possessing a high-affinity iron binding site, in a cleft between two domains. Central to function is a highly conserved set of iron ligands, including an aspartate residue (Asp63 in the N-lobe) that also hydrogen bonds between the two domains and an arginine residue (Arg124 in the N-lobe) that binds an iron-bound carbonate ion. To further probe the roles of these residues, we have determined the crystal structures of the D63E and R124A mutants of the N-terminal half-molecule of human transferrin. The structure of the D63E mutant, determined at 1.9 A resolution (R = 0.245, R(free) = 0.261), showed that the carboxyl group still binds to iron despite the larger size of the Glu side chain, with some slight rearrangement of the first turn of alpha-helix residues 63-72, to which it is attached. The structure of the R124A mutant, determined at 2.4 A resolution (R = 0.219, R(free) = 0.288), shows that the loss of the arginine side chain results in a 0.3 A displacement of the carbonate ion, and an accompanying movement of the iron atom. In both mutants, the iron coordination is changed slightly, the principal change being in each case a lengthening of the Fe-N(His249) bond. Both mutants also release iron more readily than the wild type, kinetically and in terms of acid lability of iron binding. We attribute this to more facile protonation of the synergistically bound carbonate ion, in the case of R124A, and to strain resulting from the accommodation of the larger Glu side chain, in the case of D63E. In both cases, the weakened Fe-N(His) bond may also contribute, consistent with protonation of the His ligand being an early intermediate step in iron release, following the protonation of the carbonate ion.     

Histidine-40 of ribonuclease T1 acts as base catalyst when the true catalytic base, glutamic acid-58, is replaced by alanine

Mechanisms for the ribonuclease T1 (RNase T1; EC 3.1.27.3) catalyzed transesterification reaction generally include the proposal that Glu58 and His92 provide general base and general acid assistance, respectively [Heinemann, U., & Saenger, W. (1982) Nature (London) 299, 27-31]. This view was recently challenged by the observation that mutants substituted at position 58 retain high residual activity; a revised mechanism was proposed in which His40, and not Glu58, is engaged in catalysis as general base [Nishikawa, S., Morioka, H., Kim, H., Fuchimura, K., Tanaka, T., Uesugi, S., Hakoshima, T., Tomita, K., Ohtsuka, E., & Ikehara, M. (1987) Biochemistry 26, 8620-8624]. To clarify the functional roles of His40, Glu58, and His92, we analyzed the consequences of several amino acid substitutions (His40Ala, His40Lys, His40Asp, Glu58Ala, Glu58Gln, and His92Gln) on the kinetics of GpC transesterification. The dominant effect of all mutations is on Kcat, implicating His40, Glu58, and His92 in catalysis rather than in substrate binding. Plots of log (Kcat/Km) vs pH for wild-type, His40Lys, and Glu58Ala RNase T1, together with the NMR-determined pKa values of the histidines of these enzymes, strongly support the view that Glu58-His92 acts as the base-acid couple. The curves also show that His40 is required in its protonated form for optimal activity of wild-type enzyme. We propose that the charged His40 participates in electrostatic stabilization of the transition state; the magnitude of the catalytic defect (a factor of 2000) from the His40 to Ala replacement suggests that electrostatic catalysis contributes considerably to the overall rate acceleration. For Glu58Ala RNase T1, the pH dependence of the catalytic parameters suggests an altered mechanism in which His40 and His92 act as base and acid catalyst, respectively. The ability of His40 to adopt the function of general base must account for the significant activity remaining in Glu58-mutated enzymes.     

Insight into the role of a unique SSEHA motif in the activity and stability of Helicobacter pylori arginase

Arginase is a binuclear Mn(2+) -metalloenzyme of urea cycle that hydrolyzes arginine to ornithine and urea. Unlike other arginases, the Helicobacter pylori enzyme is selective for Co(2+) and has all conserved motifs except (88) SSEHA(92) (instead of GGDHS). To examine the role of this motif in the activity and stability, steady-state kinetics, mutational analysis, thermal denaturation, and homology modeling were carried out. With a series of single and double mutants, we show that mutations of Ser88 and Ala92 to its analogous residues in other arginases individually enhance the catalytic activity. This is supported by the modeling studies, where the motif plays a role in alteration at the active site structure compared to other arginases. Mutational analysis further shows that both Glu90 and His91 are important for the activity, as their mutations lead to significant decrease in the catalytic efficiency but they appear to act in two different ways; Glu90 has a more catalytic role as its mutant displays binding of the two metal ions per monomer of the protein, but His91 plays a critical role in retaining the metal ion at the active site as its mutation exhibits a loss of one metal ion. Thermal denaturation studies demonstrated that Ser88 and His91 both play crucial roles in the stability of the protein as their mutants showed a decrease in the T(m) by ～10-11°. Unlike wild type, the metal ions have larger role in providing the stability to the mutant proteins. Thus, our data demonstrate that the motif not only plays an important role in the activity but also critical in the stability of the protein.     

Crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 at 0.95 A resolution: dynamics of catalytic residues

We present the structure of LinB, a 33-kDa haloalkane dehalogenase from Sphingomonas paucimobilis UT26, at 0.95 A resolution. The data have allowed us to directly observe the anisotropic motions of the catalytic residues. In particular, the side-chain of the catalytic nucleophile, Asp108, displays a high degree of disorder. It has been modeled in two conformations, one similar to that observed previously (conformation A) and one strained (conformation B) that approached the catalytic base (His272). The strain in conformation B was mainly in the C(alpha)-C(beta)-C(gamma) angle (126 degrees ) that deviated by 13.4 degrees from the "ideal" bond angle of 112.6 degrees. On the basis of these observations, we propose a role for the charge state of the catalytic histidine in determining the geometry of the catalytic residues. We hypothesized that double-protonation of the catalytic base (His272) reduces the distance between the side-chain of this residue and that of the Asp108. The results of molecular dynamics simulations were consistent with the structural data showing that protonation of the His272 side-chain nitrogen atoms does indeed reduce the distance between the side-chains of the residues in question, although the simulations failed to demonstrate the same degree of strain in the Asp108 C(alpha)-C(beta)-C(gamma) angle. Instead, the changes in the molecular dynamics structures were distributed over several bond and dihedral angles. Quantum mechanics calculations on LinB with 1-chloro-2,2-dimethylpropane as a substrate were performed to determine which active site conformations and protonation states were most likely to result in catalysis. It was shown that His272 singly protonated at N(delta)(1) and Asp108 in conformation A gave the most exothermic reaction (DeltaH = -22 kcal/mol). With His272 doubly protonated at N(delta)(1) and N(epsilon)(2), the reactions were only slightly exothermic or were endothermic. In all calculations starting with Asp108 in conformation B, the Asp108 C(alpha)-C(beta)-C(gamma) angle changed during the reaction and the Asp108 moved to conformation A. The results presented here indicate that the positions of the catalytic residues and charge state of the catalytic base are important for determining reaction energetics in LinB.     

A familiar motif in a new context: the catalytic mechanism of hydroxyisourate hydrolase

Hydroxyisourate hydrolase is a recently discovered enzyme that participates in the ureide pathway in soybeans. Its role is to catalyze the hydrolysis of 5-hydroxyisourate, the product of the urate oxidase reaction. There is extensive sequence homology between hydroxyisourate hydrolase and retaining glycosidases; in particular, the conserved active site glutamate residues found in retaining glycosidases are present in hydroxyisourate hydrolase as Glu 199 and Glu 408. However, experimental investigation of their roles, as well as the catalytic mechanism of the enzyme, have been precluded by the instability of 5-hydroxyisourate. Here, we report that diaminouracil serves as a slow, alternative substrate and can be used to investigate catalysis by hydroxyisourate hydrolase. The activity of the E199A protein was reduced 400-fold relative to wild-type, and no activity could be detected with the E408A mutant. Steady-state kinetic studies of the wild-type protein revealed that the pH-dependence of V(max) and V/K describe bell-shaped curves, consistent with the hypothesis that catalysis requires two ionizable groups in opposite protonation states. Addition of 100 mM azide accelerated the reaction catalyzed by the wild-type enzyme 8-fold and the E199A mutant 20-fold but had no effect on the E408A mutant. These data suggest that Glu 408 acts as a nucleophile toward the substrate forming a covalent anhydride intermediate, and Glu 199 facilitates formation of the intermediate by serving as a general acid and then activates water for hydrolysis of the intermediate. Thus, the mechanism of hydroxyisourate hydrolase is strikingly similar to that of retaining glycosidases, even though it catalyzes hydrolysis of an amide bond.     

Proton-binding sites of acid-sensing ion channel 1

Acid-sensing ion channels (ASICs) are proton-gated cation channels that exist throughout the mammalian central and peripheral nervous systems. ASIC1 is the most abundant of all the ASICs and is likely to modulate synaptic transmission. Identifying the proton-binding sites of ASCI1 is required to elucidate its pH-sensing mechanism. By using the crystal structure of ASIC1, the protonation states of each titratable site of ASIC1 were calculated by solving the Poisson-Boltzmann equation under conditions wherein the protonation states of all these sites are simultaneously in equilibrium. Four acidic-acidic residue pairs--Asp238-Asp350, Glu220-Asp408, Glu239-Asp346, and Glu80-Glu417--were found to be highly protonated. In particular, the Glu80-Glu417 pair in the inner pore was completely protonated and possessed 2 H(+), implying its possible importance as a proton-binding site. The pK(a) of Glu239, which forms a pair with a possible pH-sensing site Asp346, differs among each homo-trimer subunit due to the different H-bond pattern of Thr237 in the different protein conformations of the subunits. His74 possessed a pK(a) of ≈6-7. Conservation of His74 in the proton-sensitive ASIC3 that lacks a residue corresponding to Asp346 may suggest its possible pH-sensing role in proton-sensitive ASICs.     

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: theoretical and experimental study of the effect of glutamic acid 284 on the protonation state of lysine 213

The crystal structure of Escherichia coli phosphoenolpyruvate (PEP) carboxykinase shows Lys213 is one of the ligands of enzyme-bound Mn2+ [Nat. Struct. Biol. 4 (1997) 990]. The direct coordination of Mn2+ by N(epsilon) of Lys213 is only consistent with a neutral (uncharged) Lys213, suggesting a low pKa for this residue. This work shows, through theoretical calculations and experimental analyses on homologous Saccharomyces cerevisiae PEP carboxykinase, how the microenvironment affects Mn2+ binding and the protonation state of Lys213. We show that Glu284, a residue close to Lys212, is required for correct protonation states of Lys212 and Lys213, and for Mn2+ binding. deltaG and deltaH values for the proton reorganization processes were calculated to analyze the energetic stability of the two different protonation states of Lys212 and Lys213 in wild-type and Glu284Gln S. cerevisiae PEP carboxykinase. Calculations were done using two modeling approaches, ab-initio density functional calculations and free energy perturbation (FEP) calculations. Both methods suggest that Lys212 must be protonated and Lys213 neutral in the wild-type enzyme. On the other hand, the calculations on the Glu284Gln mutant suggest a more stable neutral Lys212 and protonated Lys213. Experimental measurements showed 3 orders of magnitude lower activity and a threefold increase in Km for Mn2+ for Glu284Gln S. cerevisiae PEP carboxykinase when compared to wild type. The data here presented suggest that Glu284 is required for Mn2+ binding by S. cerevisiae PEP carboxykinase. We propose that Glu284 modulates the pKa value of Lys213 through electrostatic effects mediated by     

ATR-FTIR study of the protonation states of the Glu residue in the multicopper oxidases, CueO and bilirubin oxidase

Redox-induced protonation state changes of the Glu residue in the multicopper oxidases, CueO and bilirubin oxidase (BO), were studied by attenuated total reflectance-Fourier transform infrared spectroscopy. By monitoring IR bands of the carboxylic acid CO stretch in the wild-type and Glu-to-Gln mutant enzymes the Glu506 of CueO (Glu463 of BO) was found to be unprotonated in the oxidised and protonated in the reduced forms. The results provided direct evidence for proton uptake by the Glu, suggesting it plays a key role in the proton donation to the activated oxygen species in the catalytic cycle.     

Biochemical characterization and identification of the catalytic residues of a family 43 beta-D-xylosidase from Geobacillus stearothermophilus T-6

Beta-D-xylosidases are hemilcellulases that hydrolyze short xylooligosaccharides into xylose units. Here, we describe the characterization and kinetic analysis of a family 43 beta-xylosidase from Geobacillus stearothermophilus T-6 (XynB3). Enzymes in this family use an inverting single-displacement mechanism with two conserved carboxylic acids, a general acid, and a general base. XynB3 was most active at 65 degrees C and pH 6.5, with clear preference to xylose-based substrates. Products analysis indicated that XynB3 is an exoglycosidase that cleaves single xylose units from the nonreducing end of xylooligomers. On the basis of sequence homology, amino acids Asp15 and Glu187 were suggested to act as the general-base and general-acid catalytic residues, respectively. Kinetic analysis with substrates bearing different leaving groups showed that, for the wild-type enzyme, the k(cat) and k(cat)/K(m) values were only marginally affected by the leaving-group reactivity, whereas for the E187G mutant, both values exhibited significantly greater dependency on the pK(a) of the leaving group. The pH-dependence activity profile of the putative general-acid mutant (E187G) revealed that the protonated catalytic residue was removed. Addition of the exogenous nucleophile azide did not affect the activities of the wild type or the E187G mutant but rescued the activity of the D15G mutant. On the basis of thin-layer chromatography and (1)H NMR analyses, xylose and not xylose azide was the only product of the accelerated reaction, suggesting that the azide ion does not attack the anomeric carbon directly but presumably activates a water molecule. Together, these results confirm the suggested catalytic role of Glu187 and Asp15 in XynB3 and provide the first unequivocal evidence regarding the exact roles of the catalytic residues in an inverting GH43 glycosidase.     

Role of water in aging of human butyrylcholinesterase inhibited by echothiophate: the crystal structure suggests two alternative mechanisms of aging

Organophosphorus poisons (OP) bind covalently to the active-site serine of cholinesterases. The inhibited enzyme can usually be reactivated with powerful nucleophiles such as oximes. However, the covalently bound OP can undergo a suicide reaction (termed aging) yielding nonreactivatable enzyme. In human butyrylcholinesterase (hBChE), aging involves the residues His438 and Glu197 that are proximal to the active-site serine (Ser198). The mechanism of aging is known in detail for the nerve gases soman, sarin, and tabun as well as the pesticide metabolite isomalathion. Aging of soman- and sarin-inhibited acetylcholinesterase occurs by C-O bond cleavage, whereas that of tabun- and isomalathion-inhibited acetylcholinesterase occurs by P-N and P-S bond cleavage, respectively. In this work, the crystal structures of hBChE inhibited by the ophthalmic reagents echothiophate (nonaged and aged) and diisopropylfluorophosphate (aged) were solved and refined to 2.1, 2.25, and 2.2 A resolution, respectively. No appreciable shift in the position of the catalytic triad histidine was observed between the aged and nonaged conjugates of hBChE. This absence of shift contrasts with the aged and nonaged crystal structures of Torpedo californica acetylcholinesterase inhibited by the nerve agent VX. The nonaged hBChE structure shows one water molecule interacting with Glu197 and the catalytic triad histidine (His438). Interestingly, this water molecule is ideally positioned to promote aging by two mechanisms: breaking either a C-O bond or a P-O bond. Pesticides and certain stereoisomers of nerve agents are expected to undergo aging by breaking the P-O bond.     

Structural analysis of the temperature-sensitive mutant of bacteriophage T4 lysozyme, glycine 156----aspartic acid

The structure of the mutant of bacteriophage T4 lysozyme in which Gly-156 is replaced by aspartic acid is described. The lysozyme was isolated by screening for temperature-sensitive mutants and has a melting temperature at pH 6.5 that is 6.1 degrees C lower than wild type. The mutant structure is destabilized, in part, because Gly-156 has conformational angles (phi, psi) that are not optimal for a residue with a beta-carbon. High resolution crystallographic refinement of the mutant structure (R = 17.7% at 1.7 A resolution) shows that the Gly----Asp substitution does not significantly alter the configurational angles (phi, psi) but forces the backbone to move, as a whole, approximately 0.6 A away from its position in wild-type lysozyme. This induced strain weakens a hydrogen bond network that exists in the wild-type structure and also contributes to the reduced stability of the mutant lysozyme. The introduction of an acidic side chain reduces the overall charge on the molecule and thereby tends to increase the stability of the mutant structure relative to wild type. However, at neutral pH this generalized electrostatic stabilization is offset by specific electrostatic repulsion between Asp-156 and Asp-92. The activity of the mutant lysozyme is approximately 50% that of wild-type lysozyme. This reduction in activity might be due to introduction of a negative charge and/or perturbation of the surface of the molecule in the region that is assumed to interact with peptidoglycan substrates.