An oxyanion-hole selective serine protease inhibitor in complex with trypsin.

p-amidinophenylmethylphosphinic acid (AMPA) was designed, synthesized and crystallized in complex with trypsin to study interactions with the oxyanion hole at the S1 site. In comparison to benzamidine, AMPA shows improved activity, which the crystal structure demonstrates to result from hydrogen bonds between the negatively charged phosphinic acid group and the catalytic residues at the oxyanion hole.     

Distinctive structural motifs co‐ordinate the catalytic nucleophile and the residues of the oxyanion hole in the alpha/beta‐hydrolase fold enzymes

The alpha/beta‐hydrolases (ABH) are among the largest structural families of proteins that are found in nature. Although they vary in their sequence and function, the ABH enzymes use a similar acid–base‐nucleophile catalytic mechanism to catalyze reactions on different substrates. Because ABH enzymes are biocatalysts with a wide range of potential applications, protein engineering has taken advantage of their catalytic versatility to develop enzymes with industrial applications. This study is a comprehensive analysis of 40 ABH enzyme families focusing on two identified substructures: the nucleophile zone and the oxyanion zone, which co‐ordinate the catalytic nucleophile and the residues of the oxyanion hole, and independently reported as critical for the enzymatic activity. We also frequently observed an aromatic cluster near the nucleophile and oxyanion zones, and opposite the ligand‐binding site. The nucleophile zone, the oxyanion zone and the residue cluster enriched in aromatic side chains comprise a three‐dimensional structural organization that shapes the active site of ABH enzymes and plays an important role in the enzymatic function by structurally stabilizing the catalytic nucleophile and the residues of the oxyanion hole. The structural data support the notion that the aromatic cluster can participate in co‐ordination of the catalytic histidine loop, and properly place the catalytic histidine next to the catalytic nucleophile.     

MD simulations reveal alternate conformations of the oxyanion hole in the Zika virus NS2B/NS3 protease

Recent crystallography studies have shown that the binding site oxyanion hole plays an important role in inhibitor binding, but can exist in two conformations (active/inactive). We have undertaken molecular dynamics (MD) calculations to better understand oxyanion hole dynamics and thermodynamics. We find that the Zika virus (ZIKV) NS2B/NS3 protease maintains a stable closed conformation over multiple 100-ns conventional MD simulations in both the presence and absence of inhibitors. The S1, S2, and S3 pockets are stable as well. However, in two of eight simulations, the A132-G133 peptide bond in the binding pocket of S1' spontaneously flips to form a 3₁₀-helix that corresponds to the inactive conformation of the oxyanion hole, and then maintains this conformation until the end of the 100-ns conventional MD simulations without inversion of the flip. This conformational change affects the S1' pocket in ZIKV NS2B/NS3 protease active site, which is important for small molecule binding. The simulation results provide evidence at the atomic level that the inactive conformation of the oxyanion hole is more favored energetically when no specific interactions are formed between substrate/inhibitor and oxyanion hole residues. Interestingly, however, transition between the active and inactive conformation of the oxyanion hole can be observed by boosting the valley potential in accelerated MD simulations. This supports a proposed induced-fit mechanism of ZIKV NS2B/NS3 protease from computational methods and provides useful direction to enhance inhibitor binding predictions in structure-based drug design.     

1H, 13C, and 15N resonance assignments of Fusarium solani pisi cutinase and preliminary features of the structure in solution.

Essentially complete (96%) sequence-specific assignments were made for the backbone and side-chain 1H, 13C, and 15N resonances of Fusarium solani pisi cutinase, produced as a 214-residue heterologous protein in Escherichia coli, using heteronuclear NMR techniques. Three structural features were noticed during the assignment. (1) The secondary structure in solution corresponds mostly with the structure from X-ray diffraction, suggesting that both structures are globally similar. (2) The HN of Ala32 has a strongly upfield-shifted resonance at 3.97 ppm, indicative of an amide-aromatic hydrogen bond to the indole ring of Trp69 that stabilizes the N-terminal side of the parallel beta-sheet. (3) The NMR data suggest that the residues constituting the oxyanion hole are quite mobile in the free enzyme in solution, in contrast to the existence of a preformed oxyanion hole as observed in the crystal structure. Apparently, cutinase forms its oxyanion hole upon binding of the substrate like true lipases.     

Crystallographic and biochemical analysis of cocaine-degrading antibody 15A10

Catalytic antibody 15A10 hydrolyzes the benzoyl ester of cocaine to form the nonpsychoactive metabolites benzoic acid and ecgonine methylester. Here, we report biochemical and structural studies that characterize the catalytic mechanism. The crystal structure of the cocaine-hydrolyzing monoclonal antibody (mAb) 15A10 has been determined at 2.35 A resolution. The binding pocket is fairly shallow and mainly hydrophobic but with a cluster of three hydrogen-bond donating residues (TrpL96, AsnH33, and TyrH35). Computational docking of the transition state analogue (TSA) indicates that these residues are appropriately positioned to coordinate the phosphonate moiety of the TSA and, hence, form an oxyanion hole. Tyrosine modification of the antibody with tetranitromethane reduced hydrolytic activity to background level. The contribution from these and other residues to catalysis and TSA binding was explored by site-directed mutagenesis of 15A10 expressed in a single chain fragment variable (scFv) format. The TyrH35Phe mutant had 4-fold reduced activity, and TrpL96Ala, TrpL96His, and AsnH33Ala mutants were all inactive. Comparison with an esterolytic antibody D2.3 revealed a similar arrangement of tryptophan, asparagine, and tyrosine residues in the oxyanion hole that stabilizes the transition state for ester hydrolysis. Furthermore, the crystal structure of the bacterial cocaine esterase (cocE) also showed that the cocE employs a tyrosine hydroxyl in the oxyanion hole. Thus, the biochemical and structural data are consistent with the catalytic antibody providing oxyanion stabilization as its major contribution to catalysis.     

Acyl-intermediate structures of the extended-spectrum class A beta-lactamase,Toho-1, in complex with cefotaxime,cephalothin, and benzylpenicillin

Bacterial resistance to beta-lactam antibiotics is a serious problem limiting current clinical therapy. The most common form of resistance is the production of beta-lactamases that inactivate beta-lactam antibiotics. Toho-1 is an extended-spectrum beta-lactamase that has acquired efficient activity not only to penicillins but also to cephalosporins including the expanded-spectrum cephalosporins that were developed to be stable in former beta-lactamases. We present the acyl-intermediate structures of Toho-1 in complex with cefotaxime (expanded-spectrum cephalosporin), cephalothin (non-expanded-spectrum cephalosporin), and benzylpenicillin at 1.8-, 2.0-, and 2.1-A resolutions, respectively. These structures reveal distinct features that can explain the ability of Toho-1 to hydrolyze expanded-spectrum cephalosporins. First, the Omega-loop of Toho-1 is displaced to avoid the steric contacts with the bulky side chain of cefotaxime. Second, the conserved residues Asn(104) and Asp(240) form unique interactions with the bulky side chain of cefotaxime to fix it tightly. Finally, the unique interaction between the conserved Ser(237) and cephalosporins probably helps to bring the beta-lactam carbonyl group to the suitable position in the oxyanion hole, thus increasing the cephalosporinase activity.     

Investigation of the induced-fit mechanism and catalytic activity of the human cytomegalovirus protease homodimer via molecular dynamics simulations

Human cytomegalovirus (HCMV) is a highly species-specific DNA virus infecting up to 80% of the general population. The viral genome contains the open reading frame UL80, which encodes the full-length 80 kDa HCMV serine protease and its substrate. Full-length HCMV protease is composed of an N-terminal 256-amino-acid proteolytic domain, called assemblin, a linker region, and a C-terminal structural domain, the assembly protein precursor. Biochemical studies have shown that dimerization activates assemblin because of an induced stabilization of the oxyanion hole (Arg166). Thus, we performed here molecular dynamics (MD) simulations on HCMV protease models to study the induced-fit mechanism of the enzyme upon the binding of substrates and peptidyl inhibitors, and structural and energetic factors that are responsible for the catalytic activity of the enzyme dimer. Long and stable trajectories were obtained for the models of the monomeric and dimeric states, free in solution and bound to a peptidyl-activated carbonyl inhibitor, with very good agreement between theoretical and experimental results. Our results suggest that HCMV protease is indeed a novel example of serine protease that operates by an induced-fit mechanism. Also, in agreement with mutagenesis studies, our MD simulations suggest that the dimeric form is necessary to activate the enzyme because of an induced stabilization of the oxyanion hole.     

Crystal Structure of Patatin-17 in Complex with Aged and Non-Aged Organophosphorus Compounds

Patatin is a non-specific plant lipase and the eponymous member of a broad class of serine hydrolases termed the patatin-like phospholipase domain containing proteins (PNPLAs). Certain PNPLA family members can be inhibited by organophosphorus (OP) compounds. Currently, no structural data are available on the modes of interaction between the PNPLAs and OP compounds or their native substrates. To this end, we present the crystal structure of patatin-17 (pat17) in its native state as well as following inhibition with methyl arachidonyl fluorophosphonate (MAFP) and inhibition/aging with diisopropylphosphorofluoridate (DFP). The native pat17 structure revealed the existence of two portals (portal1 and portal2) that lead to its active-site chamber. The DFP-inhibited enzyme underwent the aging process with the negatively charged phosphoryl oxygen, resulting from the loss of an isopropyl group, being within hydrogen-binding distance to the oxyanion hole. The MAFP-inhibited pat17 structure showed that MAFP did not age following its interaction with the nucleophilic serine residue (Ser77) of pat17 since its O-methyl group was intact. The MAFP moiety is oriented with its phosphoryl oxygen in close proximity to the oxyanion hole of pat17 and its O-methyl group located farther away from the oxyanion hole of pat17 relative to the DFP-bound state. The orientation of the alkoxy oxygens within the two OP compounds suggests a role for the oxyanion hole in stabilizing the emerging negative charge on the oxygen during the aging reaction. The arachidonic acid side chain of MAFP could be contained within portals 1 or 2. Comparisons of pat17 in the native, inhibited, and aged states showed no significant global conformational changes with respect to their Cα backbones, consistent with observations from other α/β hydrolases such as group VIIA phospholipase A2.     

A dynamic structure for the acyl-enzyme species of the antibiotic aztreonam with the Citrobacter freundii beta-lactamase revealed by infrared spectroscopy and molecular dynamics simulations

Infrared difference spectra show that at least 4 conformations coexist for the ester carbonyl group of the stable acyl-enzyme species formed between the antibiotic aztreonam and the class C beta-lactamase from Citrobacter freundii. A novel method for the assignment of the bands that arise from the ester carbonyl group has been employed. This has made use of the finding that the infrared absorption intensity of aliphatic esters is surprisingly constant, so a direct comparison with simple model esters has been possible. This has allowed a clear distinction to be made between ester and amide (protein) absorptions. The polarity of the conformer environment varies from hexane-like to strongly hydrogen-bonded. We assume that the conformer with the lowest frequency (1,690 cm(-)(1)) and hence the strongest hydrogen-bonding is the singular conformer observed in the X-ray crystallographic structure, since a good interaction via two hydrogen bonds with the oxyanion hole is seen. Molecular dynamics simulation by the method of locally enhanced sampling revealed that the motion of the ester carbonyl of the acyl-enzyme species in and out of the oxyanion hole is facile. The simulation revealed two pathways for this motion that would go through intermediates that first break one or the other of the two hydrogen bonds to the oxyanion hole, prior to departure of the carbonyl moiety out of the active site. It is likely that such motion for the acyl-enzyme species might also occur with more typical beta-lactam substrates for beta-lactamases, but their detection in the more rapid time scale may prove a challenge.     

Structural characterization of the interactions of optimized product inhibitors with the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein by NMR and modelling studies

The interactions of peptide inhibitors, obtained by the optimization of N-terminal cleavage products of natural substrates, with the protease of human hepatitis C virus (HCV) are characterized by NMR and modelling studies. The S-binding region of the enzyme and the bound conformation of the ligands are experimentally determined. The NMR data are then used as the experimental basis for modelling studies of the structure of the complex. The S-binding region involves the loop connecting strands E2 and F2, and appears shallow and solvent-exposed. The ligand binds in an extended conformation, forming an antiparallel beta-sheet with strand E2 of the protein, with the P1 carboxylate group in the oxyanion hole.     

Testing electrostatic complementarity in enzyme catalysis: hydrogen bonding in the ketosteroid isomerase oxyanion hole

A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing p K _a models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50–0.76 ppm/p K _a unit, suggesting a bond shortening of ˜0.02 Å/p K _a unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (ΔΔG = −0.2 kcal/mol/p K _a unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (ΔΔH = −2.0 kcal/mol/p K _a unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of ˜300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution, and suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from a single dominant contribution.     

Enzymatic activity of the Staphylococcus aureus SplB serine protease is induced by substrates containing the sequence Trp-Glu-Leu-Gln

Proteases are of significant importance for the virulence of Staphylococcus aureus. Nevertheless, their subset, the serine protease-like proteins, remains poorly characterized. Here presented is an investigation of SplB protease catalytic activity revealing that the enzyme possesses exquisite specificity and only cleaves efficiently after the sequence Trp-Glu-Leu-Gln. To understand the molecular basis for such selectivity, we solved the three-dimensional structure of SplB to 1.8 Å. Modeling of substrate binding to the protease demonstrated that selectivity relies in part on a canonical specificity pockets-based mechanism. Significantly, the conformation of residues that ordinarily form the oxyanion hole, an essential structural element of the catalytic machinery of serine proteases, is not canonical in the SplB structure. We postulate that within SplB, the oxyanion hole is only formed upon docking of a substrate containing the consensus sequence motif. It is suggested that this unusual activation mechanism is used in parallel with classical determinants to further limit enzyme specificity. Finally, to guide future development, we attempt to point at likely physiological substrates and thus the role of SplB in staphylococcal physiology.     

Mono- or di-substituted imidazole derivatives for inhibition of acetylcholine and butyrylcholine esterases

Mono- or di-substituted imidazole derivatives were synthesized using a one-pot, two-step strategy. All imidazole derivatives were tested for AChE and BChE inhibition and showed nanomolar activity similar to that of the test compound donepezil and higher than that of tacrine. Structure activity relationship studies, docking studies to on X-ray crystal structure of AChE with PDB code 1B41, and adsorption, distribution, metabolism, and excretion (ADME) predictions were performed. The synthesized core skeleton was bound to important regions of the active site of AChE such as the peripheral anionic site (PAS), oxyanion hole (OH), and anionic subsite (AS). Selectivity of the reported test compounds was calculated and enzyme kinetic studies revealed that they behave as competitive inhibitors, while two of the test compounds showed noncompetitive inhibitory behavior. ADME predictions revealed that the synthesized molecules might pass through the blood brain barrier and intestinal epithelial barrier and circulate freely in the blood stream without binding to human serum albumin. While the toxicity of one compound on the WS1 (skin fibroblast) cell line was 1790 µM, its toxicity on the SH-SY5Y (neuroblastoma) cell line was 950 µM.     

Crystal Structures of E. coli Native MenH and Two Active Site Mutants

Recent revision of the biosynthetic pathway for menaquinone has led to the discovery of a previously unrecognized enzyme 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase, also known as MenH. This enzyme has an α/β hydrolase fold with a catalytic triad comprising Ser86, His232, and Asp210. Mutational studies identified a number of conserved residues of importance to activity, and modeling further implicated the side chains of Tyr85 and Trp147 in formation of a non-standard oxyanion hole. We have solved the structure of E. coli MenH (EcMenH) at 2.75 Å resolution, together with the structures of the active site mutant proteins Tyr85Phe and Arg124Ala, both at 2.5 Å resolution. EcMenH has the predicted α/β hydrolase fold with its core α/β domain capped by a helical lid. The active site, a long groove beneath the cap, contains a number of conserved basic residues and is found to bind exogeneous anions, modeled as sulfate and chloride, in all three crystal structures. Docking studies with the MenH substrate and a transition state model indicate that the bound anions mark the binding sites for anionic groups on the substrate. The docking studies, and careful consideration of the active site geometry, further suggest that the oxyanion hole is of a conventional nature, involving peptide NH groups, rather than the proposed site involving Tyr85 and Trp147. This is in accord with conclusions from the structure of S. aureus MenH. Comparisons with the latter do, however, indicate differences in the periphery of the active site that could be of relevance to selective inhibition of MenH enzymes.     

Modeling evolution of hydrogen bonding and stabilization of transition states in the process of cocaine hydrolysis catalyzed by human butyrylcholinesterase

Molecular dynamics (MD) simulations and quantum mechanical/molecular mechanical (QM/MM) calculations were performed on the prereactive enzyme-substrate complex, transition states, intermediates, and product involved in the process of human butyrylcholinesterase (BChE)-catalyzed hydrolysis of (-)-cocaine. The computational results consistently reveal a unique role of the oxyanion hole (consisting of G116, G117, and A199) in BChE-catalyzed hydrolysis of cocaine, compared to acetylcholinesterase (AChE)-catalyzed hydrolysis of acetylcholine. During BChE-catalyzed hydrolysis of cocaine, only G117 has a hydrogen bond with the carbonyl oxygen (O31) of the cocaine benzoyl ester in the prereactive BChE-cocaine complex, and the NH groups of G117 and A199 are hydrogen-bonded with O31 of cocaine in all of the transition states and intermediates. Surprisingly, the NH hydrogen of G116 forms an unexpected hydrogen bond with the carboxyl group of E197 side chain and, therefore, is not available to form a hydrogen bond with O31 of cocaine in the acylation. The NH hydrogen of G116 is only partially available to form a weak hydrogen bond with O31 of cocaine in some structures involved in the deacylation. The change of the estimated hydrogen-bonding energy between the oxyanion hole and O31 of cocaine during the reaction process demonstrates how the protein environment can affect the energy barrier for each step of the BChE-catalyzed hydrolysis of cocaine. These insights concerning the effects of the oxyanion hole on the energy barriers provide valuable clues on how to rationally design BChE mutants with a higher catalytic activity for the hydrolysis of (-)-cocaine.     

Contribution of the glutamine 19 side chain to transition-state stabilization in the oxyanion hole of papain

The existence of an oxyanion hole in cysteine proteases able to stabilize a transition-state complex in a manner analogous to that found with serine proteases has been the object of controversy for many years. In papain, the side chain of Gln19 forms one of the hydrogen-bond donors in the putative oxyanion hole, and its contribution to transition-state stabilization has been evaluated by site-directed mutagenesis. Mutation of Gln19 to Ala caused a decrease in kcat/KM for hydrolysis of CBZ-Phe-Arg-MCA, which is 7700 M-1 s-1 in the mutant enzyme as compared to 464,000 M-1 s-1 in wild-type papain. With a Gln19Ser variant, the activity is even lower, with a kcat/KM value of 760 M-1 s-1. The 60- and 600-fold decreases in kcat/KM correspond to changes in free energy of catalysis of 2.4 and 3.8 kcal/mol for Gln19Ala and Gln19Ser, respectively. In both cases, the decrease in activity is in large part attributable to a decrease in kcat, while KM values are only slightly affected. These results indicate that the oxyanion hole is operational in the papain-catalyzed hydrolysis of CBZ-Phe-Arg-MCA and constitute the first direct evidence of a mechanistic requirement for oxyanion stabilization in the transition state of reactions catalyzed by cysteine proteases. The equilibrium constants Ki for inhibition of the papain mutants by the aldehyde Ac-Phe-Gly-CHO have also been determined. Contrary to the results with the substrate, mutation at position 19 of papain has a very small effect on binding of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

High resolution structures of p-aminobenzamidine- and benzamidine-VIIa/soluble tissue factor: unpredicted conformation of the 192-193 peptide bond and mapping of Ca2+, Mg2+, Na+, and Zn2+ sites in factor VIIa

Factor VIIa (FVIIa) consists of a gamma-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like domains, and a protease domain. FVIIa binds seven Ca(2+) ions in the Gla, one in the EGF1, and one in the protease domain. However, blood contains both Ca(2+) and Mg(2+), and the Ca(2+) sites in FVIIa that could be specifically occupied by Mg(2+) are unknown. Furthermore, FVIIa contains a Na(+) and two Zn(2+) sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca(2+), Mg(2+), Na(+), and Zn(2+). The crystal diffracted to 1.8A resolution, and the final structure has an R-factor of 19.8%. In this structure, the Gla domain has four Ca(2+) and three bound Mg(2+). The EGF1 domain contains one Ca(2+) site, and the protease domain contains one Ca(2+), one Na(+), and two Zn(2+) sites. (45)Ca(2+) binding in the presence/absence of Mg(2+) to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly(193) in FVIIa points away from the oxyanion hole in this structure. Importantly, the oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87A resolution. However, soaking benzamidine-FVIIa/sTF crystals with d-Phe-Pro-Arg-chloromethyl ketone results in benzamidine displacement, d-Phe-Pro-Arg incorporation, and oxyanion hole formation by a flip of the 192-193 peptide bond in FVIIa. Thus, it is the substrate and not the TF binding that induces oxyanion hole formation and functional active site geometry in FVIIa. Absence of oxyanion hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.     

A novel main-chain anion-binding site in proteins: the nest. A particular combination of phi,psi values in successive residues gives rise to anion-binding sites that occur commonly and are found often at functionally important regions.

Main-chain conformations where one amino acid residue can be described as gamma(R) (or alpha(R)) and an adjacent one as gamma(L) (or alpha(L)) mostly result in the three main-chain NH groups (of the two residues and the one following) forming a depression that can accommodate an atom with a whole or partial negative charge. We propose the name nest for this feature. The negatively charged atom, when present, is also stabilized by hydrogen-bonding with the NH groups. In an average protein, 8 % of residues are involved in a nest. The anion, or partially negatively charged atom, that often occupies the nest may be a main-chain carbonyl oxygen atom as in the paperclip, also called the Schellman loop, and the oxyanion hole of serine proteases. It can be a phosphate group, as in the P-loop superfamily that binds ATP and GTP. Overlapping, compound, nests are observed often, as in the P-loop, which has five successive NH groups that bind the beta phosphate group of nucleotide triphosphate. The longest compound nests are found surrounding cysteine-bound [2Fe2S] and [4Fe4S] iron-sulfur centers, which are also anionic; nests may encourage binding of the more reduced forms. The nest is a novel feature in the sense of not having been described as a unique motif with anion-binding potential before, although some of the situations where it occurs are familiar.     

Length of the acyl carbonyl bond in acyl-serine proteases correlates with reactivity

Resonance Raman (RR) spectroscopy has been used to obtain the vibrational spectrum of the acyl carbonyl group in a series of acylchymotrypsins and acylsubtilisins at the pH of optimum hydrolysis. The acyl-enzymes, which utilize arylacryloyl acyl groups, include three oxyanion hole mutants of subtilisin BPN', Asn155Leu, Asn155Gln, and Asn155Arg, and encompass a 500-fold range of deacylation rate constants. For each acyl-enzyme a RR carbonyl band has been identified which arises from a population of carbonyl groups undergoing nucleophilic attack in the active site. As the deacylation rate (k3) increases through the series of acyl-enzymes, the carbonyl stretching band (vC = O) is observed to shift to lower frequency, indicating an increase in single bond character of the reactive acyl carbonyl group. Experiments involving the oxyanion hole mutants of subtilisin BPN' indicate that a shift of vC = O to lower frequency results from stronger hydrogen bonding of the acyl carbonyl group in the oxyanion hole. A plot of log k3 against vC = O is linear over the range investigated, demonstrating that the changes in vC = O correlate with the free energy of activation for the deacylation reaction. By use of an empirical correlation between carbonyl frequency (vC = O) and carbonyl bond length (rC = O) it is estimated that rC = O increases by 0.015 A as the deacylation rate increases 500-fold through the series of acyl-enzymes. This change in rC = O is about 7% of that expected for going from a formal C = O double bond in the acyl-enzyme to a formal C-O single bond in the tetrahedral intermediate for deacylation. The data also allow us to estimate the energy needed to extend the acyl carbonyl group along its axis to be 950 kJ mol-1 A-1.     

High resolution crystal structures of human cytosolic thiolase (CT): a comparison of the active sites of human CT, bacterial thiolase, and bacterial KAS I

Thiolases belong to a superfamily of condensing enzymes that includes also beta-ketoacyl acyl carrier protein synthases (KAS enzymes), involved in fatty acid synthesis. Here, we describe the high resolution structure of human cytosolic acetoacetyl-CoA thiolase (CT), both unliganded (at 2.3 angstroms resolution) and in complex with CoA (at 1.6 angstroms resolution). CT catalyses the condensation of two molecules of acetyl-CoA to acetoacetyl-CoA, which is the first reaction of the metabolic pathway leading to the synthesis of cholesterol. CT is a homotetramer of exact 222 symmetry. There is an excess of positively charged residues at the interdimer surface leading towards the CoA-binding pocket, possibly important for the efficient capture of substrates. The geometry of the catalytic site, including the three catalytic residues Cys92, His 353, Cys383, and the two oxyanion holes, is highly conserved between the human and bacterial Zoogloea ramigera thiolase. In human CT, the first oxyanion hole is formed by Wat38 (stabilised by Asn321) and NE2(His353), and the second by N(Cys92) and N(Gly385). The active site of this superfamily is constructed on top of four active site loops, near Cys92, Asn321, His353, and Cys383, respectively. These loops were used for the superpositioning of CT on the bacterial thiolase and on the Escherichia coli KAS I. This comparison indicates that the two thiolase oxyanion holes also exist in KAS I at topologically equivalent positions. Interestingly, the hydrogen bonding interactions at the first oxyanion hole are different in thiolase and KAS I. In KAS I, the hydrogen bonding partners are two histidine NE2 atoms, instead of a water and a NE2 side-chain atom in thiolase. The second oxyanion hole is in both structures shaped by corresponding main chain peptide NH-groups. The possible importance of bound water molecules at the catalytic site of thiolase for the reaction mechanism is discussed.