13C nuclear magnetic resonance observations on the interaction between p-amidinophenylpyruvic acid and trypsin

The formation of an enzyme-inhibitor adduct between bovine trypsin and [2-¹³C]p-amidinophenylpyruvic acid has been investigated by ¹³C NMR spectroscopy. The observation of a resonance at 100.8 ppm demonstrates that the hemiketal formed between the hydroxyl of serine-195 and the 2-¹³C carbon of p-amidinophenylpyruvic acid is sp³ hybridized with no significant deviation from tetrahedral geometry. It is shown that stabilization of the hemiketal oxyanion if it occurs is less effective than in chloromethylketone inhibitor complexes. The tetrahedral adduct is stable from pH 3 to 8. The mechanisms of breakdown of the tetrahedral adduct at pH extremes are discussed.     

C nuclear magnetic resonance observations on the interaction between p-amidinophenylpyruvic acid and trypsin

The formation of an enzyme-inhibitor adduct between bovine trypsin and [2-¹³C]p-amidinophenylpyruvic acid has been investigated by ¹³C NMR spectroscopy. The observation of a resonance at 100.8 ppm demonstrates that the hemiketal formed between the hydroxyl of serine-195 and the 2-¹³C carbon of p-amidinophenylpyruvic acid is sp³ hybridized with no significant deviation from tetrahedral geometry. It is shown that stabilization of the hemiketal oxyanion if it occurs is less effective than in chloromethylketone inhibitor complexes. The tetrahedral adduct is stable from pH 3 to 8. The mechanisms of breakdown of the tetrahedral adduct at pH extremes are discussed.     

Quantitative structure-activity relationships for the pre-steady-state inhibition of cholesterol esterase by 4-nitrophenyl-N-substituted carbamates

4-Nitrophenyl-N-substituted carbamates (1-6) are the pseudo-substrate inhibitors of porcine pancreatic cholesterol esterase. Thus, the first step of the inhibition (Ki step) is the formation of the enzyme inhibitor tetrahedral adduct and the second step of the inhibition (kc) is the formation of the carbamyl enzyme. The formation of the enzyme inhibitor tetrahedral adduct is further divided into two steps, the formation of the enzyme-inhibitor complex with the dissociation constant, KS, at the first step and the formation of the enzyme-inhibitor tetrahedral adduct from the complex at the second step. The two-step mechanism for the formation of the enzyme-inhibitor tetrahedral adduct is confirmed by the pre-steady-state kinetics. The results of quantitative structure-activity relationships for the pre-steady-state inhibitions of cholesterol esterase by carbamates 1-6 indicate that values of -logKs and logk2/k-2 are correlated with the Taft substituent constant, sigma*, and the rho* values from these correlations are -0.33 and 0.1, respectively. The negative rho* value for the -logKS-sigma*-correlation indicates that the first step of the two-step formation of the enzyme-inhibitor tetrahedral adduct (KS step) is the formation of the positive enzyme inhibitor complex. The positive rho* value for the logk2/k-2 -sigma*-correlation indicates that the enzyme inhibitor tetrahedral adduct is more negative than the enzyme inhibitor complex. Finally, the two-step mechanism for the formation of the enzyme inhibitor tetrahedral adduct is proposed according to these results. Thus, the partially positive charge is developed at nitrogen of carbamates 1-6 in the enzyme-inhibitor complex probably due to the hydrogen bonding between the lone pair of nitrogen of carbamates 1-6 and the amide hydrogen of the oxyanion hole of the enzyme. The second step of the two-step formation of the enzyme-inhibitor tetrahedral adduct is the nucleophilic attack of the serine of the enzyme to the carbonyl group of carbamates 1-6 in the enzyme-inhibitor complex and develops the negative-charged oxygen in the adduct.     

Bisoxazaborolidines and boron complexes derived from tetradentate ligands: synthesis and spectroscopic studies

A series of tetradentate ligands were synthesized and their reaction with arylboronic acids was studied. Two classes of tetradentate ligands are discussed, which are mainly based on ONNO and ONOO donor sets and involve 2-aminophenol derivatives and Schiff bases. The reaction of phenylboronic acid with tetradentate ligands derived from 2-aminophenol leads to bisoxazaborolidines containing five-membered rings formed by CCNBO atoms, where the boron atoms have a trigonal geometry. In contrast, tetradentate ligands derived from Schiff bases lead to monomeric and dimeric boron complexes, in which the boron atoms have a tetrahedral geometry stabilized by an intramolecular N-B dative bond. The structures of two boron compounds were established by X-ray diffraction analysis.     

Aromatic boronic acids as probes of the catalytic site of human plasma lecithin-cholesterol acyltransferase

We have recently proposed a catalytic mechanism for human plasma lecithin-cholesterol acyltransferase (EC 2.3.1.43) (J. Biol. Chem. (1986) 261, 7032-7043), implicating single serine and histidine residues in phosphatidylcholine cleavage and two cysteine residues in cholesterol esterification. We now confirm the involvement of serine and histidine in catalysing the phospholipase A2 action of lecithin-cholesterol acyltransferase by demonstrating the inhibition of this activity by phenylboronic acid (Ki = 1.23 mM) and m-aminophenylboronic acid (Ki = 2.32 mM), inhibitors of known serine/histidine hydrolases. The specificity of the interaction of aromatic boronic acids with catalytic serine and histidine residues and the putative formation of a tetrahedral adduct between boron and the lecithin-cholesterol acyltransferase serine hydroxyl group which is similar to the transition-state intermediate formed between phosphatidylcholine and the catalytic serine residue was suggested by: substrate protection against inhibition by phenylboronic acids; a much reduced incorporation of phenylmethane[35S]sulphonyl fluoride into the enzyme in the presence of phenylboronic acid; the lack of interaction of histidine- or serine-modified enzyme with immobilized phenylboronic acid in the presence of glycerol (Ve/Vo = 2.7 and 2.3 respectively) when compared to the native enzyme (Ve/Vo = 5.25). Fatty acyl-lecithin-cholesterol acyltransferase, produced by incubation of the enzyme with a lecithin-apolipoprotein A-I proteoliposome substrate, was not retarded upon the sorbent column (Ve/Vo = 1.5). Modification of the enzyme's two free cysteine residues with 5,5'-dithiobis(2-nitrobenzoic acid) or potassium ferricyanide reduced (Ve/Vo = 3.5) but did not abolish retardation on the sorbent column, indicating that these modifications resulted in steric hinderance of the interaction of the boron atom with the lecithin-cholesterol acyltransferase serine hydroxyl group. These data suggest that the serine and histidine residues are proximal within the enzyme catalytic site and that both cysteine thiol groups are close to the serine hydroxyl group. The presence of significant amino-acid sequence homologies between lecithin-cholesterol acyltransferase, triacylglycerol lipases and the transacylases of fatty acid synthase is also reported.     

Boric acid and boronic acids inhibition of pigeonpea urease

Urease from the seeds of pigeonpea was competitively inhibited by boric acid, butylboronic acid, phenylboronic acid, and 4-bromophenylboronic acid; 4-bromophenylboronic acid being the strongest inhibitor, followed by boric acid > butylboronic acid > phenylboronic acid, respectively. Urease inhibition by boric acid is maximal at acidic pH (5.0) and minimal at alkaline pH (10.0), i.e., the trigonal planar B(OH)3 form is a more effective inhibitor than the tetrahedral B(OH)4 -anionic form. Similarly, the anionic form of phenylboronic acid was least inhibiting in nature.     

Quantitative structure-activity relationships for the pre-steady state of Pseudomonas species lipase inhibitions by p-nirophenyl-N-substituted carbamates

The pre-steady states of Pseudomonas species lipase inhibitions by p-nitrophenyl-N-substituted carbamates (1-6) are composed of two steps: (1) formation of the non-covalent enzyme-inhibitor complex (E:I) from the inhibitor and the enzyme and (2) formation of the tetrahedral enzyme-inhibitor adduct (E-I) from the E:I complex. From a stopped-flow apparatus, the dissociation constant for the E:I complex, KS, and the rate constant for formation of the tetrahedral E-I adduct from the E:I complex, k2 are obtained from the non-linear least-squares of curve fittings of first-order rate constant (k(obs)) versus inhibition concentration ([I]) plot against k(obs)=k2+k2[I]/(KS+[I]). Values of pKS, and log k2 are linearly correlated with the sigma* values with the rho* values of -2.0 and 0.36, respectively. Therefore, the E:I complexes are more positive charges than the inhibitors due to the rho* value of -2.0. The tetrahedral E-I adducts on the other hand are more negative charges than the E:I complexes due to the rho* value of 0.36. Formation of the E:I complex from the inhibitor and the enzyme are further divided into two steps: (1) the pre-equilibrium protonation of the inhibitor and (2) formation of the E:I complex from the protonated inhibitor and the enzyme.     

Electronic spectroscopy of cobalt angiotensin converting enzyme and its inhibitor complexes

Zinc, the catalytically essential metal of angiotensin converting enzyme (ACE), has been replaced by cobalt(II) to give an active, chromophoric enzyme that is spectroscopically responsive to inhibitor binding. Visible absorption spectroscopy and magnetic circular dichroic spectropolarimetry have been used to characterize the catalytic metal binding site in both the cobalt enzyme and in several enzyme-inhibitor complexes. The visible absorption spectrum of cobalt ACE exhibits a single broad maximum (525 nm) of relatively low absorptivity (epsilon = 75 M-1 cm-1). In contrast, the spectra of enzyme-inhibitor complexes display more clearly defined maxima at longer wavelengths (525-637 nm) and of markedly higher absorptivities (130-560 M-1 cm-1). The large spectral response indicates that changes in the cobalt ion coordination sphere occur on inhibitor binding. Magnetic circular dichroic spectropolarimetry has shown that the metal coordination geometry in the inhibitor complexes is tetrahedral and of higher symmetry than in cobalt ACE alone. The presence of sulfur----cobalt charge-transfer bands in both the visible absorption and magnetic circular dichroic spectra of the cobalt ACE-Captopril complex confirm direct ligation of the thiol group of the inhibitor to the active-site metal.     

Multinuclear magnetic resonance studies on serine protease transition state analogues

31P Nuclear Magnetic Resonance (NMR) studies were performed on mono- and diisopropylphosphoryl derivatives of alpha-chymotrypsin, trypsin, and subtilisin. Questions addressed included the pKa of the active center Asp...His...Ser triad in both species. While the pKa in the diisopropylphosphoryl derivatives is near 7.4 (found in this and other laboratories earlier) and reflects a nearly normal imidazolium titration curve, the apparent pKa in the monoisopropylphosphoryl enzymes (obtained by "aging" of the diisopropylphosphoryl derivatives and monitored by 31P NMR) is between 9.7 and 11.4 depending on the protease. This latter "titration" of the 31P NMR signal is reversible and presumably reflects the interaction of the imidazolium positive charge with the monoanionic phosphodiester. Of the two tetrahedral intermediates, the properties of the monoisopropylphosphoryl enzyme are probably more representative of the tetrahedral oxyanionic intermediate invoked during peptide hydrolysis. The same NMR technique was used to determine the action of PAM (pyridine-2-aldoxime methiodide, a known "antidote" for acetylcholinesterase inactivated by diisopropylfluorophosphate), on the inactivated enzymes. It was clear that the "antidote" could reverse the diisopropylphosphorylation but was ineffective on the monoisopropylphosphoryl ("aged") enzyme. 11B NMR studies were performed on phenylboronic (PBA) acid and 3,5-bis-trifluoromethylphenylboronic acid in the absence and presence of chymotrypsin and subtilisin. At 22 degrees C the former, but not the latter, compound was in fast exchange between the free and enzyme bound states. The relaxation parameters could be calculated for the bound PBA in chymotrypsin and the fluorinated analogue in subtilisin and clearly indicated that the boron nucleus was tetrahedral in the active centers, a good analogue for the tetrahedral oxyanionic intermediate.     

Serine protease mechanism: structure of an inhibitory complex of alpha-lytic protease and a tightly bound peptide boronic acid

The structure of the complex formed between alpha-lytic protease, a serine protease secreted by Lysobacter enzymogenes, and N-tert-butyloxycarbonylalanylprolylvaline boronic acid (Ki = 0.35 nM) has been studied by X-ray crystallography to a resolution of 2.0 A. The active-site serine forms a covalent, nearly tetrahedral adduct with the boronic acid moiety of the inhibitor. The complex is stabilized by seven hydrogen bonds between the enzyme and inhibitor with additional stabilization arising from van der Waals interactions between enzyme and inhibitor side chains and the burying of 330 A2 of hydrophobic surface area. Hydrogen bonding between Asp-102 and His-57 remains intact in the enzyme-inhibitor complex, and His N epsilon 2 is well positioned to donate its hydrogen to the leaving group. Little change in the positions of protease residues was observed on complex formation (root mean square main chain deviation = 0.13 A), suggesting that in its native state the enzyme is complementary to tetrahedral reaction intermediates or to the nearly tetrahedral transition state for the reaction.     

Mechanistic inferences from the binding of ligands to LpxC, a metal-dependent deacetylase

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

Identification of serine and histidine adducts in complexes of trypsin and trypsinogen with peptide and nonpeptide boronic acid inhibitors by 1H NMR spectroscopy.

We have previously shown, in 15N NMR studies of the enzyme's active site histidine residue, that boronic acid inhibitors can form two distinct types of complexes with alpha-lytic protease. Inhibitors that are structural analogs of good alpha-lytic protease substrates form transition-state-like tetrahedral complexes with the active site serine whereas those that are not form complexes in which N epsilon 2 of the active site histidine is covalently bonded to the boron of the inhibitor. This study also demonstrated that the serine and histidine adduct complexes exhibit quite distinctive and characteristic low-field 1H NMR spectra [Bachovchin, W. W., Wong, W. Y. L., Farr-Jones, S., Shenvi, A. B., & Kettner, C. A. (1988) Biochemistry 27, 7689-7697]. Here we have used low-field 1H NMR diagnostically for a series of boronic acid inhibitor complexes of trypsin and trypsinogen. The results show that H-D-Val-Leu-boroArg and Ac-Gly-boroArg, analogs of good trypsin substrates, form transition-state-like serine adducts with trypsin, whereas the nonsubstrate analog inhibitors boric acid, methane boronic acid, butane boronic acid, and triethanolamine borate all form histidine adducts, thereby paralleling the previous results obtained with alpha-lytic protease. However, with trypsinogen, Ac-Gly-boroArg forms predominantly a histidine adduct while H-D-Val-Leu-boroArg forms both histidine and serine adducts, with the histidine adduct predominating below pH 8.0 and the serine adduct predominating above pH 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity chromatography of yeast alpha-glucosidase using ligand-mediated chromatography on immobilized phenylboronic acids.

The synthesis of 3-nitro-4-(6-aminohexylamido)phenylboronic acid is described. The properties of two novel forms of immobilized phenylboronate agarose adsorbents [m-aminophenylboronic acid-Matrex Gel and 3-nitro-4-(6-aminohexylamido)phenylboronic acid-Sepharose CL-6B] were investigated. Both gels bind and selectively retard the glycoprotein alpha-glucosidase from yeast. The retardation is affected by following parameters: (i) pH, (ii) presence of sugar, (iii) concentration of sugar and (iv) buffer species (especially triethanolamine). Five sugars were studied, namely sorbitol, fructose, ribose, glucose and maltose. The concentration of sugar required to produce significant retardation increased in the above order, whereas the ability of sugar to form a complex with boron decreases in the same order. These effects were observed with crude as well as pure enzyme. Since alpha-glucosidase is a glycoprotein, it is proposed that this protein is mainly bound to these immobilized phenylboronates via sugar (glyco) residues. Displacement of the enzyme from the column is effected by the sugar in the buffer (or in a preincubation mixture). However, the marked pH-dependence (this retardation effect could only be observed at pH 7.4) suggests that these results are not due solely to hydrophobic or ionic mechanisms and are more complex than simple sugar-phenylboronic acid interactions.     

Rotational resonance determination of the structure of an enzyme-inhibitor complex: phosphorylation of an (aminoalkyl)phosphinate inhibitor of D-alanyl-D-alanine ligase by ATP

We have used a newly developed solid-state NMR method, rotational resonance, to establish the structure of an inhibited complex formed upon reaction of D-alanyl-D-alanine ligase, ATP, and the aminoalkyl dipeptide analogue [1(S)-aminoethyl][2-carboxy-2(R)-methyl-1- ethyl]phosphinic acid (Ib). Analogue Ib was determined to be an ATP-dependent, slow-binding inhibitor of the D-Ala-D-Ala ligase from Salmonella typhimurium, with an enzyme-inhibitor half-life of 17 days at 37 degrees C. The inhibited complex shows a 31P NMR spectrum which is very different from that which would arise from a mixture of the free inhibitor and ATP. Four well-resolved lines were observed: two (at -8 and -14 ppm) are assignable as the phosphates of ADP, the third is assignable to an inhibitor resonance (at 53 ppm) that shifts by approximately 19 ppm on binding, and the fourth is assignable to a resonance (at -3 ppm) due to a polyphosphate or phosphate ester moiety. At rotational resonance the spectrum shows evidence for strong dipolar couplings between the phosphinate phosphorus and a phosphate ester species. The dipolar coupling between the phosphorus signals at 53 and -3 ppm was measured at rotational resonance by use of numerical simulations of both the line shape of the signal and the profile of magnetization transfer between the two sites. The measured coupling, 1.0 +/- 0.2 kHz, indicates that the two species are bridged in a P-O-P linkage, with a P-P through-space distance of 2.7 +/- 0.2 A. This proves that the mechanism of inactivation involves phosphorylation of the enzyme-bound inhibitor by ATP to form a phosphoryl-phosphinate adduct.     

Stereochemical and biochemical aspects of some organoboron complexes of sulphur donor ligands

Synthetic, structural and biochemical aspects of some organoboron complexes of sulphur containing ligands having ONS and SNNS donor systems have been described. The ligands were prepared by the condensation of 1-phenyl-1,3-butanedione, 2,4-pentanedione, diphenylethanedione, 2,3-butanedione, ethanedial and 1,4-benzenedialdehyde with 2-mercaptoaniline. The unimolar reactions between phenylboronic acid and these thio-ligands have produced Ph.B (ONS) and Ph.B. (SNNS) type of biologically active complexes. These have been characterized by elemental analysis, molecular weight determinations, and conductivity measurements. Based on UV, IR, 1H NMR, 13C NMR and 11B NMR spectral studies, a tetracoordinated state of boron has been established in all the derivatives. The ligands and their corresponding organoboron complexes have been tested in vitro against a number of pathogenic fungi and bacteria and found to possess remarkable fungicidal and bactericidal properties.     

Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. Crystal structure determination and stereochemistry of the contact region

The structure of the complex of bovine trypsin and bovine pancreatic trypsin inhibitor has been determined by crystal structure analysis at 2.8 Å resolution. The structure is closely similar to the model predicted from the structures of the components. The complex is a tetrahedral adduct with a covalent bond between the carbonyl carbon of Lys-15I of the inhibitor and the γ-oxygen of Ser-195 of the enzyme. The imidazole of His-57 is hydrogen-bonded to Asp-102 and the bound seryl γ-oxygen in accord with the histidine being charged. The negatively charged carbonyl oxygen of Lys-15I forms two hydrogen bonds with the amide nitrogens of Gly-193 and Ser-195. Protonation of the leaving group N-H of Ala-16I to form an acyl-complex requires a conformational change of the imidazole of His-57. The tetrahedral adduct is further stabilized by hydrogen bonds between groups at the leaving group side and inhibitor and enzyme, which would be weakened in the acyl-enzyme. The kinetic data of inhibitor-enzyme interaction are reconciled with the structural model, and relations between enzyme-inhibitor interaction and productive enzyme-substrate interaction are proposed.     

The investigation of recognition interaction between phenylboronate monolayer and glycated hemoglobin using surface plasmon resonance

Glycated hemoglobin (HbA1c) is formed by a nonenzymatic reaction of glucose with the N-terminal valine of adult hemoglobin's beta-chain. The amount of HbA1c reflects the average concentration of glucose variation level over the preceding 2 to 3 months. Because the boronate has antibody mimicking for HbA1c, often it is used to detect HbA1c. However, factors such as the ratio of the phenylboronic acid derivatives and diol composition, the pH of the solution, and the stereostructure of phenylboronic acid derivatives could influence the interactions between phenylboronic acid derivatives and diol composition. In this study, the factors were evaluated using surface plasmon resonance (SPR). The results show that pH value is an important factor affecting HbA1c and phenylboronic acid to form the complex and Lewis bases. This could change the stereostructure of phenylboronic acid to form B(OH)(3) for binding with saccharine easily. In addition, linear response appeared in HbA1c in the range of 0.43 to 3.49 mug/ml, and the detection limit was 0.01 microg/ml. The results also demonstrated that an SPR biosensor can be used as a sensitive technique for improving the accuracy and correctness of HbA1c measurement.     

A study of the reaction of 2-phenyl-1,3,2-benzodiazaborolidine with benzoyl chloride

Reaction of 2-phenyl-1,3,2-benzodiazaborolidine (2) with benzoyl chloride afforded N,N′-dibenzoyl-o-phenylenediamine (8) regardless of the amount of the acid chloride employed. Depending on the work-up procedure, the 2-phenylboron moiety was isolated as either phenylboronic acid or phenylboronic acid anhydride. In the absence of added bases, the reaction was observed to be susceptible to solvent assistance by ethers, presumably by coordination with the empty p-orbital of boron. In the presence of added amines, the extent of benzoylation was altered considerably. With pyridine as a co-reagent, small amounts of 1,3-dibenzoyl-2-phenyl-1,3,2,-benzodiazaborolidine mono- or dihydrocholoride were isolated though this compound readily underwent hydrolysis or oxidation upon standing. Interaction of 2 with sodium hydride in tetrahydrofuran caused ionization of only one of the two hydrogens bound to nitrogen even upon extended reflux. Benzoylation of the heterocycle under these latter conditions again afforded only the dibenzoylated compound 8.     

Structural Basis for Bacterial Quorum Sensing-mediated Oxalogenesis

The Burkholderia species utilize acetyl-CoA and oxaloacetate, substrates for citrate synthase in the TCA cycle, to produce oxalic acid in response to bacterial cell to cell communication, called quorum sensing. Quorum sensing-mediated oxalogenesis via a sequential reaction by ObcA and ObcB counteracts the population-collapsing alkaline pH of the stationary growth phase. Thus, the oxalic acid produced plays an essential role as an excreted public good for survival of the group. Here, we report structural and functional analyses of ObcA, revealing mechanistic features distinct from those of citrate synthase. ObcA exhibits a unique fold, in which a (β/α)₈-barrel fold is located in the C-domain with the N-domain inserted into a loop following α1 in the barrel fold. Structural analyses of the complexes with oxaloacetate and with a bisubstrate adduct indicate that each of the oxaloacetate and acetyl-CoA substrates is bound to an independent site near the metal coordination shell in the barrel fold. In catalysis, oxaloacetate serves as a nucleophile by forming an enolate intermediate mediated by Tyr³²² as a general base, which then attacks the thioester carbonyl carbon of acetyl-CoA to yield a tetrahedral adduct between the two substrates. Therefore, ObcA catalyzes its reaction by combining the enolase and acetyltransferase superfamilies, but the presence of the metal coordination shell and the absence of general acid(s) produces an unusual tetrahedral CoA adduct as a stable product. These results provide the structural basis for understanding the first step in oxalogenesis and constitute an example of the functional diversity of an enzyme for survival and adaptation in the environment.     

A Combined Quantum/Classical Molecular Dynamics Study of the Catalytic Mechanism of HIV Protease

Based on available three-dimensional structures of enzyme-inhibitor complexes, the mechanism of the reaction catalysed by HIV protease is studied using molecular dynamics simulations with molecular mechanics and combined quantum-mechanics/molecular-mechanics potential energy functions. The results support the general acid/general base catalysis mechanism, with Asp25′ protonated in the enzyme-substrate complex. In the enzyme-substrate complex, the lytic water molecule binds at a position different from the positions of the hydroxyl groups in various aspartic protease-inhibitor complexes. The carboxyl groups at the active site also adopt a different orientation. However, when the lytic water molecule approaches the scissile peptide, the reaction centre changes gradually to a conformation close to that derived from X-ray diffraction studies of various enzyme-inhibitor complexes. The proton transfer processes can take place only after the lytic water molecule has approached the scissile peptide bond to a certain degree. Qualitatively, the free-energy barrier associated with the nucleophilic attack step, which takes place at physiological pH, is comparable with the acid or base-catalysed reactions of model systems. The structure of the tetrahedral intermediate resulting from the nucleophilic attack step also indicates a straightforward pathway of the next reaction step, i.e. the breaking of the C-N bond.