Enzyme-product Complex Research Articles

1.2Å-resolution crystal structures reveal the second tetrahedral intermediates of streptogrisin B (SGPB)

Streptogrisin B (SGPB) has served as one of the models for studying the catalytic activities of serine peptidases. Here we report its native crystal structures at pH 4.2 at a resolution of 1.18Å, and at pH 7.3 at a resolution of 1.23Å. Unexpectedly, outstanding electron density peaks occurred in the active site and the substrate-binding region of SGPB in the computed maps at both pHs. The densities at pH 4.2 were assigned as a tetrapeptide, Asp-Ala-Ile-Tyr, whereas those at pH 7.3 were assigned as a tyrosine molecule and a leucine molecule existing at equal occupancies in both of the SGPB molecules in the asymmetric unit. Refinement with relaxed geometric restraints resulted in molecular structures representing mixtures of the second tetrahedral intermediates and the enzyme-product complexes of SGPB existing in a pH-dependent equilibrium. Structural comparisons with the complexes of SGPB with turkey ovomucoid third domain (OMTKY3) and its variants have shown that, upon the formation of the tetrahedral intermediate, residues Glu192A to Gly193 of SGPB move towards the α-carboxylate O of residue P1 of the bound species, and adjustments in the side-chain conformational angles of His57 and Ser195 of SGPB favor the progression of the catalytic mechanism of SGPB.

Revisiting the mechanism of the autoactivation of the complement protease C1r in the C1 complex: Structure of the active catalytic region of C1r

C1r is a modular serine protease which is the autoactivating component of the C1 complex of the classical pathway of the complement system. We have determined the first crystal structure of the entire active catalytic region of human C1r. This fragment contains the C-terminal serine protease (SP) domain and the preceding two complement control protein (CCP) modules. The activated CCP1–CCP2–SP fragment makes up a dimer in a head-to-tail fashion similarly to the previously characterized zymogen. The present structure shows an increased number of stabilizing interactions. Moreover, in the crystal lattice there is an enzyme–product relationship between the C1r molecules of neighboring dimers. This enzyme–product complex exhibits the crucial S1–P1 salt bridge between Asp631 and Arg446 residues, and intermolecular interaction between the CCP2 module and the SP domain. Based on these novel structural information we propose a new split-and-reassembly model for the autoactivation of the C1r. This model is consistent with experimental results that have not been explained adequately by previous models. It allows autoactivation of C1r without large-scale, directed movement of C1q arms. The model is concordant with the stability of the C1 complex during activation of the next complement components.

Kinetic Mechanism of Human dUTPase, an Essential Nucleotide Pyrophosphatase Enzyme

Human dUTPase is essential in controlling relative cellular levels of dTTP/dUTP, both of which can be incorporated into DNA. The nuclear isoform of the enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies. The recently determined three-dimensional structure of this protein in complex with an isosteric substrate analogue allowed in-depth structural characterization of the active site. However, fundamental steps of the dUTPase enzymatic cycle have not yet been revealed. This knowledge is indispensable for a functional understanding of the molecular mechanism and can also contribute to the design of potential antagonists. Here we present detailed pre-steady-state and steady-state kinetic investigations using a single tryptophan fluorophore engineered into the active site of human dUTPase. This sensor allowed distinction of the apoenzyme, enzyme-substrate, and enzyme-product complexes. We show that the dUTP hydrolysis cycle consists of at least four distinct enzymatic steps: (i) fast substrate binding, (ii) isomerization of the enzyme-substrate complex into the catalytically competent conformation, (iii) a hydrolysis (chemical) step, and (iv) rapid, nonordered release of the products. Independent quenched-flow experiments indicate that the chemical step is the rate-limiting step of the enzymatic cycle. To follow the reaction in the quenched-flow, we devised a novel method to synthesize gamma-(32)P-labeled dUTP. We also determined by indicator-based rapid kinetic assays that proton release is concomitant with the rate-limiting hydrolysis step. Our results led to a quantitative kinetic model of the human dUTPase catalytic cycle and to direct assessment of relative flexibilities of the C-terminal arm, critical for enzyme activity, in the enzyme-ligand complexes along the reaction pathway.

A Cyclobutanone Analogue Mimics Penicillin in Binding to Isopenicillin N Synthase

A carbocyclic analogue of the beta-lactam antibiotic isopenicillin N (IPN) has been synthesised and cocrystallised with isopenicillin N synthase (IPNS), the central enzyme in the biosynthesis of penicillin antibiotics. The crystal structure of the IPNS-cyclobutanone complex reveals an active site environment similar to that seen in the enzyme-product complex generated by turnover of the natural substrate within the crystalline protein. The IPNS-cyclobutanone structure demonstrates that the product analogue is tethered to the protein by hydrogen bonding and salt bridge interactions with its carboxylate groups, as seen previously for the natural substrate and product. Furthermore, the successful cocrystallisation of this analogue with IPNS provides firm structural evidence for the utility of such cyclobutanone derivatives as hydrolytically stable analogues of bicyclic beta-lactams.

Probing the Reaction Mechanism of the d-ala-d-ala Dipeptidase, VanX, by Using Stopped-Flow Kinetic and Rapid-Freeze Quench EPR Studies on the Co(II)-Substituted Enzyme

In an effort to probe the reaction mechanism of VanX, the d-ala-d-ala dipeptidase required for high-level vancomycin resistance in bacteria, stopped-flow kinetic and rapid-freeze quench EPR studies were conducted on the Co(II)-substituted enzyme when reacted with d-ala-d-ala. The intensity of the Co(II) ligand field band at 550 nm decreased (epsilon550 = 140 to 18 M-1 cm-1) when VanX was reacted with substrate, suggesting that the coordination number of the metal increases from 5 to 6 upon substrate binding. The stopped-flow trace was fitted to a kinetic mechanism that suggests the presence of an intermediate whose breakdown is rate-limiting. Rapid-freeze quench EPR studies verified the presence of a reaction intermediate that exhibits an unusually low hyperfine constant (33 G), which suggests a bidentate coordination of the intermediate to the metal center. The EPR studies also identified a distinct enzyme product complex. The results were used to offer a detailed reaction mechanism for VanX that can be used to guide future inhibitor design efforts.

X-ray, NMR, and Mutational Studies of the Catalytic Cycle of the GDP-Mannose Mannosyl Hydrolase Reaction,

GDP-mannose hydrolase catalyzes the hydrolysis with inversion of GDP-alpha-D-hexose to GDP and beta-D-hexose by nucleophilic substitution by water at C1 of the sugar. Two new crystal structures (free enzyme and enzyme-substrate complex), NMR, and site-directed mutagenesis data, combined with the structure of the enzyme-product complex reported earlier, suggest a four-stage catalytic cycle. An important loop (L6, residues 119-125) contains a ligand to the essential Mg2+ (Gln-123), the catalytic base (His-124), and three anionic residues. This loop is not ordered in the X-ray structure of the free enzyme due to dynamic disorder, as indicated by the two-dimensional 1H-15N HMQC spectrum, which shows selective exchange broadening of the imidazole nitrogen resonances of His-124 (k(ex) = 6.6 x 10(4) s(-1)). The structure of the enzyme-Mg2+-GDP-mannose substrate complex of the less active Y103F mutant shows loop L6 in an open conformation, while the structure of the enzyme-Mg2+-GDP product complex showed loop L6 in a closed, "active" conformation. 1H-15N HMQC spectra show the imidazole N epsilon of His-124 to be unprotonated, appropriate for general base catalysis. Substituting Mg2+ with the more electrophilic metal ions Mn2+ or Co2+ decreases the pKa in the pH versus kcat rate profiles, showing that deprotonation of a metal-bound water is partially rate-limiting. The H124Q mutation, which decreases kcat 10(3.4)-fold and largely abolishes its pH dependence, is rescued by the Y103F mutation, which increases kcat 23-fold and restores its pH dependence. The structural basis of the rescue is the fact that the Y103F mutation shifts the conformational equilibrium to the open form moving loop L6 out of the active site, thus permitting direct access of the specific base hydroxide from the solvent. In the proposed dissociative transition state, which occurs in the closed, active conformation of the enzyme, the partial negative charge of the GDP leaving group is compensated by the Mg2+, and by the closing of loop L2 that brings Arg-37 closer to the beta-phosphate. The development of a positive charge at mannosyl C1, as the oxocarbenium-like transition state is approached, is compensated by closing the anionic loop, L6, onto the active site, further stabilizing the transition state.

Kinetic and chemical analyses of the cytokinin dehydrogenase-catalysed reaction: correlations with the crystal structure.

CKX (cytokinin dehydrogenase) is a flavoprotein that cleaves cytokinins to adenine and the corresponding side-chain aldehyde using a quinone-type electron acceptor. In the present study, reactions of maize (Zea mays) CKX with five different substrates (N6-isopentenyladenine, trans-zeatin, kinetin, p-topolin and N-methyl-isopentenyladenine) were studied. By using stopped-flow analysis of the reductive half-reaction, spectral intermediates were observed indicative of the transient formation of a binary enzyme-product complex between the cytokinin imine and the reduced enzyme. The reduction rate was high for isoprenoid cytokinins that showed formation of a charge-transfer complex of reduced enzyme with bound cytokinin imine. For the other cytokinins, flavin reduction was slow and no charge-transfer intermediates were observed. The binary complex of reduced enzyme and imine product intermediate decays relatively slowly to form an unbound product, cytokinin imine, which accumulates in the reaction mixture. The imine product only very slowly hydrolyses to adenine and an aldehyde derived from the cytokinin N6 side-chain. Mixing of the substrate-reduced enzyme with Cu2+/imidazole as an electron acceptor to monitor the oxidative half-reaction revealed a high rate of electron transfer for this type of electron acceptor when using N6-isopentenyladenine. The stability of the cytokinin imine products allowed their fragmentation analysis and structure assessment by Q-TOF (quadrupole-time-of-flight) MS/MS. Correlations of the kinetic data with the known crystal structure are discussed for reactions with different cytokinins.

Metallo-β-lactamases: Novel Weaponry for Antibiotic Resistance in Bacteria

Metallo-beta-lactamases are broad-spectrum zinc enzymes, able to inactivate most clinically useful beta-lactam antibiotics. Their structural and functional diversity has thus far limited the understanding of their catalytic mechanism, therefore thwarting the rational design of a common inhibitor. On the basis of the recent availability of structures of enzyme-product complexes and novel mechanistic studies, here, we attempt to find minimal common elements in different members of this family. In contrast with other metalloenzymes, most of the substrate binding and catalytic power resides in the adequate positioning of one or two Zn(II) ions in the active site, empowered by an unusual flexibility.

Applications of isothermal titration calorimetry to measure enzyme kinetics and activity in complex solutions

The application of isothermal titration calorimetry (ITC) was tested towards measurements of enzyme kinetics in complex solutions containing high concentrations of proteins. Such investigations are important, due to the increasing interest in biochemical reactions in physiological relevant media as well as the application of enzymes in industrial processes. In contrast to spectral methods, measurements performed with ITC, are independent of the optical properties of solutions, making it possible to measure enzyme kinetics in concentrated solutions of macromolecules. In this study the kinetic properties of hexokinase was investigated in concentrated protein solutions (BSA). It was found, that the quality of the measured kinetic data was independent of protein concentration in the investigated range (0–250 mg BSA ml −1). All results could be accounted for by Michaelis–Menten's approach and both k cat and K M decreased with increasing protein concentrations. The decrease in K M with increasing protein concentration was ascribed to an increase in the ratio of activity coefficients between the native enzyme and the enzyme–substrate complex. The decrease in k cat with increasing protein concentrations indicates that crowding by BSA effect the conformational changes/rehydration that accompanies catalysis and/or diffusion of product from the enzyme–product complex. The methodology is discussed together with an analysis of the experimental results.

Crystal Structure of Heparinase II from Pedobacter heparinus and Its Complex with a Disaccharide Product

Heparinase II depolymerizes heparin and heparan sulfate glycosaminoglycans, yielding unsaturated oligosaccharide products through an elimination degradation mechanism. This enzyme cleaves the oligosaccharide chain on the nonreducing end of either glucuronic or iduronic acid, sharing this characteristic with a chondroitin ABC lyase. We have determined the first structure of a heparin-degrading lyase, that of heparinase II from Pedobacter heparinus (formerly Flavobacterium heparinum), in a ligand-free state at 2.15 A resolution and in complex with a disaccharide product of heparin degradation at 2.30 A resolution. The protein is composed of three domains: an N-terminal alpha-helical domain, a central two-layered beta-sheet domain, and a C-terminal domain forming a two-layered beta-sheet. Heparinase II shows overall structural similarities to the polysaccharide lyase family 8 (PL8) enzymes chondroitin AC lyase and hyaluronate lyase. In contrast to PL8 enzymes, however, heparinase II forms stable dimers, with the two active sites formed independently within each monomer. The structure of the N-terminal domain of heparinase II is also similar to that of alginate lyases from the PL5 family. A Zn2+ ion is bound within the central domain and plays an essential structural role in the stabilization of a loop forming one wall of the substrate-binding site. The disaccharide binds in a long, deep canyon formed at the top of the N-terminal domain and by loops extending from the central domain. Based on structural comparison with the lyases from the PL5 and PL8 families having bound substrates or products, the disaccharide found in heparinase II occupies the "+1" and "+2" subsites. The structure of the enzyme-product complex, combined with data from previously characterized mutations, allows us to propose a putative chemical mechanism of heparin and heparan-sulfate degradation.

Enzyme Dynamics along the Reaction Coordinate: Critical Role of a Conserved Residue

Conformational flexibility of the enzyme architecture is essential for biological function. These structural transitions often encompass significant portions of the enzyme molecule. Here, we present a detailed study of functionally relevant RNase A dynamics in the wild type and a D121A mutant form by NMR spin-relaxation techniques. In the wild-type enzyme, the dynamic properties are largely conserved in the apo, enzyme-substrate, and enzyme-product complexes. In comparison, mutation of aspartic acid 121 to alanine disrupts the timing of active-site dynamics, the product-release step, and global conformational changes, indicating that D121 plays a significant role in coordinating the dynamic events in RNase A. In addition, this mutation results in 90% loss of catalytic activity despite the absence of direct participation of D121 in the chemical reaction or in interactions with the substrate. These data suggest that one role of this conserved residue is to facilitate important millisecond protein dynamics.

Transient State Kinetic Studies of the MutT-Catalyzed Nucleoside Triphosphate Pyrophosphohydrolase Reaction

The MutT pyrophosphohydrolase, in the presence of Mg2+, catalyzes the hydrolysis of nucleoside triphosphates by nucleophilic substitution at Pbeta, to yield the nucleotide and PP(i). The best substrate for MutT is the mutagenic 8-oxo-dGTP, on the basis of its Km being 540-fold lower than that of dGTP. Product inhibition studies have led to a proposed uni-bi-iso kinetic mechanism, in which PP(i) dissociates first from the enzyme-product complex (k3), followed by NMP (k4), leaving a product-binding form of the enzyme (F) which converts to the substrate-binding form (E) in a partially rate-limiting step (k5) [Saraswat, V., et al. (2002) Biochemistry 41, 15566-15577]. Single- and multiple-turnover kinetic studies of the hydrolysis of dGTP and 8-oxo-dGTP and global fitting of the data to this mechanism have yielded all of the nine rate constants. Consistent with an "iso" mechanism, single-turnover studies with dGTP and 8-oxo-dGTP hydrolysis showed slow apparent second-order rate constants for substrate binding similar to their kcat/Km values, but well below the diffusion limit (approximately 10(9) M(-1) s(-1)): k(on)app = 7.2 x 10(4) M(-1) s(-1) for dGTP and k(on)app = 2.8 x 10(7) M(-1) s(-1) for 8-oxo-dGTP. These low k(on)app values are fitted by assuming a slow iso step (k5 = 12.1 s(-1)) followed by fast rate constants for substrate binding: k1 = 1.9 x 10(6) M(-1) s(-1) for dGTP and k1 = 0.75 x 10(9) M(-1) s(-1) for 8-oxo-dGTP (the latter near the diffusion limit). With dGTP as the substrate, replacing Mg2+ with Mn2+ does not change k1, consistent with the formation of a second-sphere MutT-M2+-(H2O)-dGTP complex, but slows the iso step (k5) 5.8-fold, and its reverse (k(-5)) 25-fold, suggesting that the iso step involves a change in metal coordination, likely the dissociation of Glu-53 from the enzyme-bound metal so that it can function as the general base. Multiple-turnover studies with dGTP and 8-oxo-dGTP show bursts of product formation, indicating partially rate-limiting steps following the chemical step (k2). With dGTP, the slow steps are the chemical step (k2 = 10.7 s(-1)) and the iso step (k5 = 12.1 s(-1)). With 8-oxo-dGTP, the slow steps are the release of the 8-oxo-dGMP product (k4 = 3.9 s(-1)) and the iso step (k5 = 12.1 s(-1)), while the chemical step is fast (k2 = 32.3 s(-1)). The transient kinetic studies are generally consistent with the steady state kcat and Km values. Comparison of rate constants and free energy diagrams indicate that 8-oxo-dGTP, at low concentrations, is a better substrate than dGTP because it binds to MutT 395-fold faster, dissociates 46-fold slower, and has a 3.0-fold faster chemical step. The true dissociation constants (KD) of the substrates from the E-form of MutT, which can now be obtained from k(-1)/k1, are 3.5 nM for 8-oxo-dGTP and 62 microM for dGTP, indicating that 8-oxo-dGTP binds 1.8 x 10(4)-fold tighter than dGTP, corresponding to a 5.8 kcal/mol lower free energy of binding.

Crystal Structure of Truncated Fibrobacter succinogenes 1,3-1,4-β- d-Glucanase in Complex with β-1,3-1,4-Cellotriose

Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fsbeta-glucanase) catalyzes the specific hydrolysis of beta-1,4 glycosidic bonds adjacent to beta-1,3 linkages in beta-D-glucans or lichenan. This is the first report to elucidate the crystal structure of a truncated Fsbeta-glucanase (TFsbeta-glucanase) in complex with beta-1,3-1,4-cellotriose, a major product of the enzyme reaction. The crystal structures, at a resolution of 2.3 angstroms, reveal that the overall fold of TFsbeta-glucanase remains virtually unchanged upon sugar binding. The enzyme accommodates five glucose residues, forming a concave active cleft. The beta-1,3-1,4-cellotriose with subsites -3 to -1 bound to the active cleft of TFsbeta-glucanase with its reducing end subsite -1 close to the key catalytic residues Glu56 and Glu60. All three subsites of the beta-1,3-1,4-cellotriose adopted a relaxed C(1)4 conformation, with a beta-1,3 glycosidic linkage between subsites -2 and -1, and a beta-1,4 glycosidic linkage between subsites -3 and -2. On the basis of the enzyme-product complex structure observed in this study, a catalytic mechanism and substrate binding conformation of the active site of TFsbeta-glucanase is proposed.

Structural Snapshots of MTA/AdoHcy Nucleosidase Along the Reaction Coordinate Provide Insights into Enzyme and Nucleoside Flexibility During Catalysis

MTA/AdoHcy nucleosidase (MTAN) irreversibly hydrolyzes the N9-C1' bond in the nucleosides, 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (AdoHcy) to form adenine and the corresponding thioribose. MTAN plays a vital role in metabolic pathways involving methionine recycling, biological methylation, polyamine biosynthesis, and quorum sensing. Crystal structures of a wild-type (WT) MTAN complexed with glycerol, and mutant-enzyme and mutant-product complexes have been determined at 2.0A, 2.0A, and 2.1A resolution, respectively. The WT MTAN-glycerol structure provides a purine-free model and in combination with the previously solved thioribose-free MTAN-ADE structure, we now have separate apo structures for both MTAN binding subsites. The purine and thioribose-free states reveal an extensive enzyme-immobilized water network in their respective binding subsites. The Asp197Asn MTAN-MTA and Glu12Gln MTAN-MTR.ADE structures are the first enzyme-substrate and enzyme-product complexes reported for MTAN, respectively. These structures provide representative snapshots along the reaction coordinate and allow insight into the conformational changes of the enzyme and the nucleoside substrate. A "catalytic movie" detailing substrate binding, catalysis, and product release is presented.

The structure of an enzyme–product complex reveals the critical role of a terminal hydroxide nucleophile in the bacterial phosphotriesterase mechanism

A detailed understanding of the catalytic mechanism of enzymes is an important step toward improving their activity for use in biotechnology. In this paper, crystal soaking experiments and X-ray crystallography were used to analyse the mechanism of the Agrobacterium radiobacter phosphotriesterase, OpdA, an enzyme capable of detoxifying a broad range of organophosphate pesticides. The structures of OpdA complexed with ethylene glycol and the product of dimethoate hydrolysis, dimethyl thiophosphate, provide new details of the catalytic mechanism. These structures suggest that the attacking nucleophile is a terminally bound hydroxide, consistent with the catalytic mechanism of other binuclear metallophosphoesterases. In addition, a crystal structure with the potential substrate trimethyl phosphate bound non-productively demonstrates the importance of the active site cavity in orienting the substrate into an approximation of the transition state.

Identification of base-specific contacts in protein–DNA complexes by photocrosslinking and mass spectrometry: a case study using the restriction endonuclease SsoII

Specific protein-nucleic acid interactions are of paramount importance for the propagation, maintenance and expression of genetic information. Restriction endonucleases serve as model systems to study the mechanisms of DNA recognition by proteins. SsoII is a Type II restriction endonuclease that recognizes the double stranded sequence downward arrow CCNGG and cleaves it in the presence of Mg(2+)-ions, as indicated. SsoII shows sequence similarity over a stretch of approximately 70 amino acid residues with several other restriction endonucleases that recognize a similar sequence as SsoII (Cfr10I, EcoRII, NgoMIV, PspGI). In NgoMIV this stretch is involved in DNA recognition and cleavage, as shown by the crystal structure analysis of an enzyme-product complex. To find out whether the presumptive DNA recognition region in SsoII is indeed in contact with DNA we have photocrosslinked SsoII with an oligodeoxyribonucleotide in which the first guanine of the recognition sequence was replaced by 5-iodouracil. Following digestion by trypsin, the peptide-oligodeoxyribonucleotide conjugate was purified by Fe(3+)-IMAC and then incubated with hydrogen fluoride, which hydrolyzes the oligodeoxyribonucleotide to yield the peptide-deoxyuridine conjugate. The site of photocrosslinking was identified by MALDI-TOF-MS and MALDI-TOF-MS/MS to be Trp189, adjacent to Arg188, which aligns with Arg194 in NgoMIV, involved in recognition of the second guanine in the NgoMIV recognition sequence G downward arrow CCGGC. This result confirms previously published conclusions drawn on the basis of a mutational analysis of SsoII. The methodology that was employed here can be used in principle to identify the DNA binding site of any protein.

On the Catalytic Role of the Conserved Active Site Residue His466 of Choline Oxidase

The oxidation of alcohols to aldehydes is catalyzed by a number of flavin-dependent enzymes, which have been grouped in the glucose-methanol-choline oxidoreductase enzyme superfamily. These enzymes exhibit little sequence similarity in their substrates binding domains, but share a highly conserved catalytic site, suggesting a similar activation mechanism for the oxidation of their substrates. In this study, the fully conserved histidine residue at position 466 of choline oxidase was replaced with an alanine residue by site-directed mutagenesis and the biochemical, spectroscopic, and mechanistic properties of the resulting CHO-H466A mutant enzyme were characterized. CHO-H466A showed k(cat) and k(cat)/K(m) values with choline as substrate that were 60- and 1000-fold lower than the values for the wild-type enzyme, while the k(cat)/K(m) value for oxygen was unaffected, suggesting the involvement of His(466) in the oxidation of the alcohol substrate but not in the reduction of oxygen. Replacement of His(466) with alanine significantly affected the microenvironment of the flavin, as indicated by the altered behavior of CHO-H466A with sulfite and dithionite. In agreement with this conclusion, a midpoint reduction potential of +106 mV for the two-electron transfer in the catalytically competent enzyme-product complex was determined at pH 7 for CHO-H466A, which was approximately 25 mV more negative than that of the wild-type enzyme. Enzymatic activity in CHO-H466A could be partially rescued with exogenous imidazolium, but not imidazole, consistent with the protonated form of histidine exerting a catalytic role. pH profiles for glycine betaine inhibition, the deprotonation of the N(3)-flavin locus, and the k(cat)/K(m) value for choline all showed a significant shift upward in their pK(a) values, consistent with a change in the polarity of the active site. Finally, kinetic isotope effects with isotopically labeled substrate and solvent indicated that the histidine to alanine substitution affected the timing of substrate OH and CH bond cleavages, consistent with removal of the hydroxyl proton being concerted with hydride transfer in the mutant enzyme. All taken together, the results presented in this study suggest that in choline oxidase, His(466) modulates the electrophilicity of the enzyme-bound flavin and the polarity of the active site, and contributes to the stabilization of the transition state for the oxidation of choline to betaine aldehyde.

Investigation of zinc-containing peptide deformylase fromLeptospira interrogansby X-ray absorption near-edge spectroscopy

Peptide deformylase (PDF, EC 3.5.1.27) is essential for the normal growth of eubacterium but not for mammalians. Recently, PDF has been studied as a target for new antibiotics. Its activity is strongly dependent on the bound metal ion. The crystallographic studies did not show any significant structural difference upon various bound metal ions. In this paper, X-ray absorption spectroscopy was employed to determine the local structure around the zinc ion of PDF from Leptospira interrogans in dry powder. XANES (X-ray absorption near-edge structure) calculations were performed and the local geometry of the active center was reconstructed successfully. By comparing with the crystal structure of an enzyme-product complex, the results from calculations show that a water molecule has moved towards the zinc ion and lies in the distance range to coordinate with the zinc ion weakly.

Structure, function and interfacial allosterism in phospholipase A2: insight from the anion-assisted dimer

Enzymes that function on membrane surfaces offer many challenges to understanding structural and functional details due to the difficulties of obtaining relevant information of the protein in a physiological environment. Focusing on this aspect of structural biology, it is important to develop conditions that mimic the interaction of membrane proteins with their binding surface and ultimately the mechanisms of action. This approach has been used to characterize the allosteric nature of secreted phospholipase A2 (PLA2) to its substrate interface. The breakthrough here was to crystallize the pancreatic group-IB PLA2 in an anion-assisted dimer with five coplanar phosphate anions bound. In the anion-assisted dimer structure one molecule of a tetrahedral mimic inhibitor and five anions are shared between the two subunits of the dimer. The sn-2-phosphate of the inhibitor, which mimics the tetrahedral intermediate of the esterolysis reaction, is bound in the active site of one subunit, and the alkyl chain extends into the active site slot of the second subunit across the subunit–subunit interface. This interface-bound structural mimic provided insight into the active site environment and specific anionic interactions to the i-face of the protein. The presence or absence of a single critical active site water, corresponds to the difference between the activated or inactivated form of the enzyme. The anion-assisted dimer structure supports a calcium coordinated nucleophilic water mechanism, with its p K a modulated by this assisting water. This working model has been further strengthened with an enzyme–product complex structure solved with the hydrolysis products of the substrate PAF also bound to the anion-assisted dimer form of PLA2. Additional confirmation of the assisting-water mechanism comes from a structure of the inactive zymogen proPLA2 also crystallized in an anion-assisted dimer. Remarkably, the assisting water present in the activated complex is absent in this proPLA2 structure.

Crystallographic snapshots of Aspergillus fumigatus phytase, revealing its enzymatic dynamics.

Understanding of the atomic movements involved in an enzymatic reaction needs structural information on the active and inactive native enzyme molecules and on the enzyme-substrate, enzyme-intermediate, and enzyme-product(s) complexes. By using the X-ray crystallographic method, four crystal structures of Aspergillus fumigatus phytase were obtained at resolution higher than 1.7 A. The pH-dependent catalytic activity of A. fumigatus phytase was linked to three water molecules that may prevent the substrate from binding and thus block nucleophilic attack of the catalytic imidazole nitrogen. Comparison of various structures also identified the water molecule that attacks the phosphamide bond during the hydrolysis process, and established the hydrolysis pathway of the intermediate. Additionally, two reaction product phosphates were observed at the active site, suggesting a possible product release pathway after hydrolysis of the intermediate. These results can help explain the catalytic mechanism throughout the whole acid phosphatase family, as all key residues are conserved.