Rapid Equilibrium Random Kinetic Mechanism Research Articles

Allosteric Inhibition of Acinetobacter baumannii ATP Phosphoribosyltransferase by Protein:Dipeptide and Protein:Protein Interactions.

ATP phosphoribosyltransferase (ATPPRT) catalyzes the first step of histidine biosynthesis in bacteria, namely, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N1-(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate. Catalytic (HisGS) and regulatory (HisZ) subunits assemble in a hetero-octamer where HisZ activates HisGS and mediates allosteric inhibition by histidine. In Acinetobacter baumannnii, HisGS is necessary for the bacterium to persist in the lung during pneumonia. Inhibition of ATPPRT is thus a promising strategy for specific antibiotic development. Here, A. baumannii ATPPRT is shown to follow a rapid equilibrium random kinetic mechanism, unlike any other ATPPRT. Histidine noncompetitively inhibits ATPPRT. Binding kinetics indicates histidine binds to free ATPPRT and to ATPPRT:PRPP and ATPPRT:ATP binary complexes with similar affinity following a two-step binding mechanism, but with distinct kinetic partition of the initial enzyme:inhibitor complex. The dipeptide histidine-proline inhibits ATPPRT competitively and likely uncompetitively, respectively, against PRPP and ATP. Rapid kinetics analysis shows His-Pro binds to the ATPPRT:ATP complex via a two-step binding mechanism. A related HisZ that shares 43% sequence identity with A. baumannii HisZ is a tight-binding allosteric inhibitor of A. baumannii HisGS. These findings lay the foundation for inhibitor design against A. baumannii ATPPRT.

The Wall Teichoic Acid Polymerase TagF Is Non-processive in Vitro and Amenable to Study Using Steady State Kinetic Analysis

Wall teichoic acids are a chemically diverse group of anionic polymers that constitute up to 50% of the Gram-positive cell wall. These polymers play a pivotal role in virulence and have been implicated in a diverse range of physiological functions. The TagF-like family of enzymes has been shown to be responsible for wall teichoic acid priming and polymerization events. Although many such enzymes are well validated therapeutic targets, a mechanistic understanding of this enzyme family has remained elusive. TagF is the prototypical teichoic acid polymerase and uses CDP-glycerol to catalyze synthesis of the linear (1,3)-linked poly(glycerol phosphate) teichoic acid in Bacillus subtilis 168. Here we used a synthetic soluble analog of the natural substrate of the enzyme, Lipid , to conduct the first detailed mechanistic investigation of teichoic acid polymerization. Through the use of a new high pressure liquid chromatography-based assay to monitor single glycerol phosphate incorporations into the Lipid analog, we conducted a detailed analysis of reaction product formation patterns and unequivocally showed TagF to be non-processive in vitro. Furthermore by monitoring the kinetics of polymerization, we showed that Lipid analog species varying in size have the same K(m) value of 2.6 microm and validated use of Bi Bi velocity expressions to model the TagF enzyme system. Initial rate analysis showed that TagF catalyzes a sequential Bi Bi mechanism where both substrates are added to the enzyme prior to product release consistent with a single displacement chemical mechanism.

Importance in Catalysis of the 6-Phosphate-binding Site of 6-Phosphogluconate in Sheep Liver 6-Phosphogluconate Dehydrogenase

The 6-phosphate of 6-phosphogluconate (6PG) is proposed to anchor the sugar phosphate in the active site and aid in orientating the substrate for catalysis. In order to test this hypothesis, alanine mutagenesis was used to probe the contribution of residues in the vicinity of the 6-phosphate to binding of 6PG and catalysis. The crystal structure of sheep liver 6-phosphogluconate dehydrogenase shows that Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 contribute a mixture of ionic and hydrogen bonding interactions to the 6-phosphate, and these interactions are likely to provide the majority of the binding energy for 6PG. All mutant enzymes, with the exception of T262A, exhibit an increase in K(6PG) that ranges from 5- to 800-fold. There is also a less pronounced increase in K(NADP), ranging from 3- to 15-fold, with the exception of T262A. The R287A and R446A mutant enzymes exhibit a dramatic decrease in V/E(t) (600- and 300-fold, respectively) as well as in V/K(6PG)E(t) (10(5) - and 10(4)-fold), and therefore no further characterization was carried out with these two mutant enzymes. No change in V/E(t) was observed for the Y191A mutant enzyme, whereas 20- and 3-fold decreases were obtained for the K260A and T262A mutant enzymes, respectively, resulting in a decrease in V/K(6PG)E(t) range from 3- to 120-fold. All mutant enzymes also exhibit at least an order of magnitude increase in 13C-isotope effect -1, indicating that the decarboxylation step has become more rate-limiting. Data are consistent with significant roles for Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 in providing the binding energy for 6PG. In addition, these residues also likely ensure proper orientation of 6PG for catalysis and aid in inducing the conformation change that precedes, and sets up the active site for, catalysis.

Nucleotide Complexes of Escherichia coli Phosphoribosylaminoimidazole Succinocarboxamide Synthetase

Phosphoribosylaminoimidazole-succinocarboxamide synthetase (SAICAR synthetase) converts 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) to 4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide (SAICAR). The enzyme is a target of natural products that impair cell growth. Reported here are the crystal structures of the ADP and the ADP.CAIR complexes of SAICAR synthetase from Escherichia coli, the latter being the first instance of a CAIR-ligated SAICAR synthetase. ADP and CAIR bind to the active site in association with three Mg(2+), two of which coordinate the same oxygen atom of the 4-carboxyl group of CAIR; whereas, the third coordinates the alpha- and beta-phosphoryl groups of ADP. The ADP.CAIR complex is the basis for a transition state model of a phosphoryl transfer reaction involving CAIR and ATP, but also supports an alternative chemical pathway in which the nucleophilic attack of l-aspartate precedes the phosphoryl transfer reaction. The polypeptide fold for residues 204-221 of the E. coli structure differs significantly from those of the ligand-free SAICAR synthetase from Thermatoga maritima and the adenine nucleotide complexes of the synthetase from Saccharomyces cerevisiae. Conformational differences between the E. coli, T. maritima, and yeast synthetases suggest the possibility of selective inhibition of de novo purine nucleotide biosynthesis in microbial organisms.

Enoyl-ACP Reductase (FabI) of Haemophilus influenzae: Steady-State Kinetic Mechanism and Inhibition by Triclosan and Hexachlorophene

Steady-state kinetics, equilibrium binding, and primary substrate kinetic isotope effect studies revealed that the reduction of crotonyl-CoA by NADH, catalyzed by Haemophilus influenzae enoyl-ACP reductase (FabI), follows a rapid equilibrium random kinetic mechanism with negative interaction among the substrates. Two biphenyl inhibitors, triclosan and hexachlorophene, were studied in the context of the kinetic mechanism. IC50 values for triclosan in the presence and absence of NAD+ were 0.1 ± 0.02 and 2.4 ± 0.02 μM, respectively, confirming previous observations that the E–NAD+ complex binds triclosan more tightly than the free enzyme. Preincubation of the enzyme with triclosan and NADH suggested that the E–NADH complex is the active triclosan binding species as well. These results were reinforced by measurement of binding kinetic transients. Intrinsic protein fluorescence changes induced by binding of 20 μM triclosan to E, E–NADH, E–NAD+, and E–crotonyl-CoA occur at rates of 0.0124 ± 0.001, 0.0663 ± 0.002, 0.412 ± 0.01, and 0.0069 ± 0.0001 s−1, respectively. The rate of binding decreased with increasing crotonyl-CoA concentrations in the E-crotonyl-CoA complex, and the extrapolated rate at zero concentration of crotonyl-CoA corresponded to the rate observed for the binding to the free enzyme. This suggests that triclosan and the acyl substrate share a common binding site. Hexachlorophene inhibition, on the other hand, was NAD+- and time-independent; and the calculated IC50 value was 2.5 ± 0.4 μM. Steady-state inhibition patterns did not allow the mode of inhibition to be unambiguously determined, but binding kinetics suggested that free enzyme, E–NAD+, and E–crotonyl-CoA have similar affinity for hexachlorophene, since the kobss were in the same range of 20–24 s−1. When the E–NADH complex was mixed with hexachlorophene ligand, concentration-independent fluorescence quenching at 480 nm was observed, suggesting at least partial competition between NADH and hexachlorophene for the same binding site. Mutual exclusivity studies, together with the above-discussed results, indicate that triclosan and hexachlorophene bind at different sites of H. influenzae FabI.

The allosteric transition of glucosamine-6-phosphate deaminase: the structure of the T state at 2.3 A resolution.

The allosteric hexameric enzyme glucosamine-6-phosphate deaminase from Escherichia coli catalyses the regulatory step of N-acetylglucosamine catabolism, which consists of the isomerisation and deamination of glucosamine 6-phosphate (GlcN6P) to form fructose 6-phosphate (Fru6P) and ammonia. The reversibility of the catalysis and its rapid-equilibrium random kinetic mechanism, among other properties, make this enzyme a good model for studying allosteric processes. Here we present the structure of P6(3)22 crystals, obtained in sodium acetate, of GlcN6P deaminase in its ligand-free T state. These crystals are very sensitive to X-ray radiation and have a high (78%) solvent content. The activesite lid (residues 162-185) is highly disordered in the T conformer; this may contribute significantly to the free-energy change of the whole allosteric transition. Comparison of the structure with the crystallographic coordinates of the R conformer (Brookhaven Protein Data Bank entry 1 dea) allows us to describe the geometrical changes associated with the allosteric transition as the movement of two rigid entities within each monomer. The active site, located in a deep cleft between these two rigid entities, presents a more open geometry in the T conformer than in the R conformer. The differences in active-site geometry are related to alterations in the substrate-binding properties associated with the allosteric transition. The rigid nature of the two mobile structural units of each monomer seems to be essential in order to explain the observed kinetics of the deaminase hexamer. The triggers for both the homotropic and heterotropic allosteric transitions are discussed and particular residues are assigned to these functions. A structural basis for an entropic term in the allosteric transition is an interesting new feature that emerges from this study.

Kinetic and Chemical Mechanisms of the Sheep Liver 6-Phosphogluconate Dehydrogenase

A complete kinetic characterization of sheep liver 6-phosphogluconate dehydrogenase including product and dead-end inhibition patterns, primary deuterium isotope effects, and the pH dependence of kinetic parameters has been completed in order to determine the kinetic mechanism and obtain information on the chemical mechanism of the enzyme. A rapid equilibrium random kinetic mechanism has been proposed, with product and dead-end inhibition patterns both being symmetric. Ribulose 5-phosphate and 6-sulfogluconate are both competitive with 6-phosphogluconate (6-PG) and noncompetitive with NADP, and NADPH and ATP-ribose are both competitive with NADP and noncompetitive with 6-phosphogluconate. Equal primary deuterium isotope effects of 1.5–2 onDV,DV/KNADP, andDV/K6-PGwith 3-deuterio-6-PG confirm a rapid equilibrium random mechanism and show that hydride transfer is at least partially rate limiting in the overall reaction. The maximum velocity is pH dependent, decreasing at low and high pH with slopes of 1 and −1, respectively, and pKvalues of 6.4 and 8.6. TheV/KNADPandV/K6-PGalso decrease at low and high pH with slopes of 1 and −1, giving pKvalues of 6.8 and 8.7 and of 6.9 and 7.8, respectively. The pH rate profiles are consistent with a general acid/general base mechanism where the catalytic residues are involved in binding. Reverse protonation states between the general acid and the general base are proposed where an unprotonated general base accepts a proton from the C-3 hydroxyl of 6-PG concomitant with hydride transfer followed by decarboxylation of the resulting 3-keto intermediate to give an enediol which is protonated by the general acid to form ribulose 5-phosphate. The pH dependence of the pKiprofile of the inhibitory analog 5-phosphoribonate decreases at low and high pH with slopes of 1 and −1, respectively, and pKs of 6.2 and 7.4 and suggests that intrinsic pKs are observed in theV/Kprofiles. The pKs of both the general base and general acid in the E:6-PG complex appears to be perturbed such that the general base decreases from 7.4–7.8 to a value of 6.4–6.8, and the pKof the general acid increases from 6.2–6.9 to a value of 8.6–8.7, as a result of direct interaction with 6PG. Data are interpreted with regard to the published crystal structures of the E:6-PG, E:NADP, and E:NADPH complexes.

Purification and Characterization of Dihydropyrimidine Dehydrogenase fromAlcaligenes eutrophus

Dihydropyrimidine dehydrogenase fromAlcaligenes eutrophuswas purified to homogeneity using ammonium sulfate fractionation and chromatography on phenyl-Sepharose, MonoQ-Sepharose, and 2,5-ADP-Sepharose. The enzyme is a homotetramer with a subunit molecular mass of 52 kDa. The absorption spectrum of the bacterial dihydropyrimidine dehydrogenase has maxima in the 300- and 400-nm region, suggesting a flavoprotein. The enzyme contains 4 mol FMN, about 24 mol iron and acid-labile sulfide per mole of protein, implying a flavoprotein with FeS centers. The bacterial dehydrogenase is NADPH dependent with B-side stereospecificity. The initial velocity patterns of the bacterial dehydrogenase together with isotope exchange at equilibrium and a quantitative analysis of the product and dead-end inhibition data suggest a rapid equilibrium random kinetic mechanism, which is in contrast to results obtained for dihydropyrimidine dehydrogenase from pig liver. The pig liver enzyme adheres to a nonclassical two-site ping-pong kinetic mechanism [B. Podschun, P. F. Cook, and K. D. Schnackerz (1990)J. Biol. Chem.265, 12966–12972], whereas for the bovine enzyme a rapid equilibrium random kinetic mechanism was proposed based on steady-state kinetic data [D. J. T. Porter and T. Spector (1993)J. Biol. Chem.268, 19321–19327].

Overall kinetic mechanism of 6-phosphogluconate dehydrogenase from Candida utilis

A complete initial velocity study of the 6-phosphogluconate dehydrogenase from Candida utilis at pH 7 and 25 degrees C in both reaction directions suggests a rapid equilibrium random kinetic mechanism with dead-end E:NADP:(ribulose 5-phosphate) and E:NADPH:(6-phosphogluconate) complexes. Like substrate-product (NADP/NADPH and 6-phosphogluconate/ribulose 5-phosphate) pairs are competitive whatever the concentration of the other substrates but noncompetitive versus the other substrates, e.g., NADPH exhibits noncompetitive inhibition versus 6-phosphogluconate. This trend also holds true for all dead-end analogs, e.g., ATP-ribose is competitive versus NADP and noncompetitive versus 6-phosphogluconate. A quantitative analysis of the kinetic inhibition constants supports the assignment of kinetic mechanism. The ratio of the maximum velocities in the oxidative decarboxylation and reductive carboxylation directions is 75.

Transition-state analysis of nucleoside hydrolase from Crithidia fasciculata

The transition state of nucleoside hydrolase from the trypanosome Crithidia fasciculata has been characterized by multiple Vmax/Km kinetic isotope effects with labeled inosine and adenosine as substrates. Nucleoside hydrolase catalyzes the hydrolysis of the N-glycosidic linkage of the commonly occurring purine and pyrimidine nucleosides, with Vmax/Km ranging over 2 orders of magnitude. The kinetic isotope effects for inosine were [1'-3H] = 1.150 +/- 0.006, [2'-3H] = 1.161 +/- 0.003, [1'-14C] = 1.044 +/- 0.004, [9-15N] = 1.026 +/- 0.004, [4'-3H] = 0.992 +/- 0.003, and [5'-3H] = 1.051 +/- 0.003. The magnitude of the kinetic isotope effects for inosine, an equivalent [1'-3H] kinetic isotope effect for the poor substrate adenosine, and the rapid equilibrium random kinetic mechanism [Parkin D, W., Horenstein, B. A., Abdulah, D. R., Estupiñán, B., & Schramm, V. L. (1991) J. Biol. Chem. (in press)] all indicate that the isotope effects are fully expressed. The kinetic and solvent deuterium isotope effects have been used to analyze the transition-state structure using bond energy bond order vibrational analysis. The transition state involves a protonated hypoxanthine leaving group with a C-N glycosidic bond elongated to approximately 2 A. The ribose group contains substantial carbocationic character, unusually strong hyperconjugation of H2', and a bond length of approximately 3 A to the incoming oxygen nucleophile. The remote isotope effect (4'-3H and 5'-3H) and the results of transition-state calculations provide the most detailed description of the steric and bonding properties of an enzyme-stabilized transition state.

AMP Deaminase from Yeast: Role in amp degradation, large scale purification, and properties of the native and proteolyzed enzyme

Eukaryotes have been proposed to depend on AMP deaminase as a primary step in the regulation of intracellular adenine nucleotide pools. This report describes 1) the role of AMP deaminase in adenylate metabolism in yeast cell extracts, 2) a method for large scale purification of the enzyme, 3) the kinetic properties of native and proteolyzed enzymes, 4) the kinetic reaction mechanism, and 5) regulatory interactions with ATP, GTP, MgATP, ADP, and PO4. Allosteric regulation of yeast AMP deaminase is of physiological significance, since expression of the gene is constitutive (Meyer, S. L., Kvalnes-Krick, K. L., and Schramm, V. L. (1989) Biochemistry 28, 8734-8743). The metabolism of ATP in cell-free extracts of yeast demonstrates that AMP deaminase is the sole pathway of AMP catabolism in these extracts. Purification of the enzyme from bakers' yeast yields a proteolytically cleaved enzyme, Mr 86,000, which is missing 192 amino acids from the N-terminal region. Extracts of Escherichia coli containing a plasmid with the gene for yeast AMP deaminase contained only the unproteolyzed enzyme, Mr 100,000. The unproteolyzed enzyme is highly unstable during purification. Substrate saturation plots for proteolyzed AMP deaminase are sigmoidal. In the presence of ATP, the allosteric activator, the enzyme exhibits normal saturation kinetics. ATP activates the proteolyzed AMP deaminase by increasing the affinity for AMP from 1.3 to 0.2 mM without affecting VM. Activation by ATP is more efficient than MgATP, with half-maximum activation constants of 6 and 80 microM, respectively. The kinetic properties of the proteolyzed and unproteolyzed AMP deaminase are similar. Thus, the N-terminal region is not required for catalysis or allosteric activation. AMP deaminase is competitively inhibited by GTP and PO4 with respect to AMP. The inhibition constants for these inhibitors decrease in the presence of ATP. ATP, therefore, tightens the binding of GTP, PO4, and AMP. The products of the reaction, NH3 and IMP, are competitive inhibitors against substrate, consistent with a rapid equilibrium random kinetic mechanism. Kinetic dissociation constants are reported for the binary and ternary substrate and product complexes and the allosteric modulators.

Isotope exchange as a probe of the kinetic mechanism of pyrophosphate-dependent phosphofructokinase.

Data obtained from isotope exchange at equilibrium, exchange of inorganic phosphate against forward reaction flux, and positional isotope exchange of 18O from the bridge position of pyrophosphate to a nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii has a rapid equilibrium random kinetic mechanism. The maximum rates of isotope exchange at equilibrium for the [14C]fructose 1,6-bisphosphate in equilibrium fructose 6-phosphate, [32P]Pi in equilibrium MgPPi, and Mg[32P]PPi in equilibrium fructose 1,6-bisphosphate exchange reactions increasing all four possible substrate-product pairs in constant ratio are identical, consistent with a rapid equilibrium mechanism. All exchange reactions are strongly inhibited at high concentrations of the fructose 6-phosphate (F6P)/Pi and MgPPi/Pi substrate-product pairs and weakly inhibited at high concentrations of the MgPPi/fructose 1,6-bisphosphate (FBP) pair suggesting three dead-end complexes, E:F6P:Pi, E:MgPPi:Pi, and E:FBP:MgPPi, in agreement with initial velocity studies [Bertagnolli, B.L., & Cook, P.F. (1984) Biochemistry 23, 4101]. Neither back-exchange by [32P]Pi nor positional isotope exchange of 18O-bridge-labeled pyrophosphate was observed under any conditions, suggesting that either the chemical interconversion step or a step prior to it limits the overall rate of the reaction.

Mechanistic aspects of the low-molecular-weight phosphatase activity of the calmodulin-activated phosphatase, calcineurin.

Product and substrate analogs have been employed as inhibitors of the low-molecular-weight phosphatase activity of calcineurin, a calmodulin-activated protein phosphatase. Product inhibition kinetics demonstrate that both products, para-nitrophenol and inorganic phosphate, inhibit para-nitrophenyl phosphate hydrolysis in a competitive manner. Inorganic phosphate is a linear competitive inhibitor, whereas the inhibition by para-nitrophenol is more complex. An analog of para-nitrophenol, pentafluorophenol, was found to be a linear competitive inhibitor. These patterns indicate a rapid equilibrium random kinetic mechanism for calcineurin. This mechanism suggests that calcineurin does not generate a phosphoryl enzyme during its catalytic reaction. Application of sulfate analogs indicates that binding of substrate occurs via the phosphoryl moiety. It is suggested that binding is a function of the affinity of ligand for the metal ion involved in calcineurin action. The dependence of the kinetic parameters of calcineurin upon pH was examined to provide information concerning the role of protonation in the activity and specificity of calcineurin. Log (VM) versus pH data for two low-molecular-weight substrates, para-nitrophenyl phosphate and tyrosine-O-phosphate, reveal a pKa value for the enzyme-substrate complex. Analysis of log (VM/KM) data yields a pKa value for the free enzyme of 8.0. Protonation of the phenolic leaving group during hydrolysis is not the rate-limiting step in calcineurin catalysis.

Enzymatic properties of the beta-galactoside alpha 1 leads to 2 fucosyltransferase from porcine submaxillary gland.

The acceptor substrate specificity and kinetic properties of the purified porcine submaxillary beta-galactoside alpha 1 leads to 2 fucosyltransferase have been examined. The transferase forms the Fuc alpha 1 leads to 2Gal linkage with oligosaccharides, glycoproteins, and glycolipids which contain nonreducing terminal galactose residues and shows no absolute specificity for a particular penultimate residue or for the linkage between the galactose and the penultimate residue. The fucosyltransferase is active in the absence of divalent metal ions, but it is stimulated upon addition of Mn2+, Mg2+, Ca2+, or Co2+. Kinetic analysis indicates an increase in the Km for both donor and acceptor substrates and in the Vmax in the presence of Mn2+. Initial rate studies and inhibition patterns suggest that the transferase has either a rapid equilibrium random kinetic mechanism or a steady state ordered mechanism with GDP-fucose binding first. Human "Bombay" erythrocytes which lack cell surface Fuc alpha 1 leads to 2Gal structures are fucosylated by the transferase, but expression of H blood group activity is dependent on treatment of the cells with neuraminidase. After neuraminidase digestion, the fucosylated cells are serologically identical to native O-type cells. Analysis of the fucosylated material in the erythrocyte membrane on sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggests that fucose is incorporated primarily into glycoprotein acceptors.

Kinetic and structural properties of Nad-Dependent bacterial formate dehydrogenase

A kinetic analysis of NAD-dependent formate dehydrogenase from methylotrophic bacteria, strain no. 1, has been carried out. Initial velocity, product, and dead-end inhibition studies are compatible with the rapid-equilibrium random kinetic mechanism. Chemical modification studies of formate dehydrogenase reveal essential cysteine and arginine residues. The influence of enzyme inactivation on the modification of hystidine and lysine residues was also investigated. The model of the enzyme active centre is proposed. Some practical applications of formate dehydrogenase are discussed.