Chorismate synthase. Pre-steady-state kinetics of phosphate release from 5-enolpyruvylshikimate 3-phosphate.
Ontology highlight
ABSTRACT:
SUBMITTER: Hawkes TR
PROVIDER: S-EPMC1133717 | biostudies-other | 1990 Feb
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The first step of histidine biosynthesis in Acinetobacter baumannii, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate to produce N1-(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate, is catalyzed by the hetero-octameric enzyme ATP phosphoribosyltransferase, a promising target for antibiotic design. The catalytic subunit, HisGS, is allosterically activated upon binding of the regulatory subunit, HisZ, to form the hetero-octameric holoenzyme (ATPPRT), leading to a large increase in kcat. Here, we present the crystal structure of ATPPRT, along with kinetic investigations of the rate-limiting steps governing catalysis in the nonactivated (HisGS) and activated (ATPPRT) forms of the enzyme. A pH-rate profile showed that maximum catalysis is achieved above pH 8.0. Surprisingly, at 25 °C, kcat is higher when ADP replaces ATP as substrate for ATPPRT but not for HisGS. The HisGS-catalyzed reaction is limited by the chemical step, as suggested by the enhancement of kcat when Mg2+ was replaced by Mn2+, and by the lack of a pre-steady-state burst of product formation. Conversely, the ATPPRT-catalyzed reaction rate is determined by PRATP diffusion from the active site, as gleaned from a substantial solvent viscosity effect. A burst of product formation could be inferred from pre-steady-state kinetics, but the first turnover was too fast to be directly observed. Lowering the temperature to 5 °C allowed observation of the PRATP formation burst by ATPPRT. At this temperature, the single-turnover rate constant was significantly higher than kcat, providing additional evidence for a step after chemistry limiting catalysis by ATPPRT. This demonstrates allosteric activation by HisZ accelerates the chemical step.
| S-EPMC10795190 | biostudies-literature
Project description:The transient kinetic behavior of enzyme reactions prior to the establishment of steady state is a major source of mechanistic information, yet this approach has not been utilized for cellulases acting on their natural substrate, insoluble cellulose. Here, we elucidate the pre-steady-state regime for the exo-acting cellulase Cel7A using amperometric biosensors and an explicit model for processive hydrolysis of cellulose. This analysis allows the identification of a pseudo-steady-state period and quantification of a processivity number as well as rate constants for the formation of a threaded enzyme complex, processive hydrolysis, and dissociation, respectively. These kinetic parameters elucidate limiting factors in the cellulolytic process. We concluded, for example, that Cel7A cleaves about four glycosidic bonds/s during processive hydrolysis. However, the results suggest that stalling the processive movement and low off-rates result in a specific activity at pseudo-steady state that is 10-25-fold lower. It follows that the dissociation of the enzyme-substrate complex (half-time of ~30 s) is rate-limiting for the investigated system. We suggest that this approach can be useful in attempts to unveil fundamental reasons for the distinctive variability in hydrolytic activity found in different cellulase-substrate systems.
| S-EPMC3365755 | biostudies-literature
Project description:DNA polymerase fidelity is defined as the ratio of right (R) to wrong (W) nucleotide incorporations when dRTP and dWTP substrates compete at equal concentrations for primer extension at the same site in the polymerase-primer-template DNA complex. Typically, R incorporation is favored over W by 10(3)-10(5)-fold, even in the absence of 3'-exonuclease proofreading. Straightforward in principle, a direct competition fidelity measurement is difficult to perform in practice because detection of a small amount of W is masked by a large amount of R. As an alternative, enzyme kinetics measurements to evaluate k(cat)/K(m) for R and W in separate reactions are widely used to measure polymerase fidelity indirectly, based on a steady state derivation by Fersht. A systematic comparison between direct competition and kinetics has not been made until now. By separating R and W products using electrophoresis, we have successfully taken accurate fidelity measurements for directly competing R and W dNTP substrates for 9 of the 12 natural base mispairs. We compare our direct competition results with steady state and pre-steady state kinetic measurements of fidelity at the same template site, using the proofreading-deficient mutant of Klenow fragment (KF(-)) DNA polymerase. All the data are in quantitative agreement.
| S-EPMC2803633 | biostudies-literature
Project description:Cruzain, an important drug target for Chagas disease, is a member of clan CA of the cysteine proteases. Understanding the catalytic mechanism of cruzain is vital to the design of new inhibitors. To this end, we have determined pH-rate profiles for substrates and affinity agents and solvent kinetic isotope effects in pre-steady-state and steady-state modes using three substrates: Cbz-Phe-Arg-AMC, Cbz-Arg-Arg-AMC, and Cbz-Arg-Ala-AMC. The pH-rate profile of kcat/ Km for Cbz-Arg-Arg-AMC indicated p K1 = 6.6 (unprotonated) and p K2 ∼ 9.6 (protonated) groups were required for catalysis. The temperature dependence of the p K = 6.2-6.6 group exhibited a Δ Hion value of 8.4 kcal/mol, typical of histidine. The pH-rate profile of inactivation by iodoacetamide confirmed that the catalytic cysteine possesses a p Ka of 9.8. Normal solvent kinetic isotope effects were observed for both D2O kcat (1.6-2.1) and D2O kcat/ Km (1.1-1.4) for all three substrates. Pre-steady-state kinetics revealed exponential bursts of AMC production for Cbz-Phe-Arg-AMC and Cbz-Arg-Arg-AMC, but not for Cbz-Arg-Ala-AMC. The overall solvent isotope effect on kcat can be attributed to the solvent isotope effect on the deacylation step. Our results suggest that cruzain is unique among papain-like cysteine proteases in that the catalytic cysteine and histidine have neutral charges in the free enzyme. The generation of the active thiolate of the catalytic cysteine is likely preceded (and possibly triggered) by a ligand-induced conformational change, which could bring the catalytic dyad into the proximity to effect proton transfer.
| S-EPMC10569748 | biostudies-literature
Project description:Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a homotetrameric protein with one tungstodipterin and one [4Fe-4S] cubane per 69-kDa subunit. The enzyme kinetics have been studied under steady-state conditions at 80 degrees C and pre-steady state conditions at 50 degrees C, in the latter case via monitoring of the relatively weak (epsilon approximately 2 mM(-1) cm(-1)) optical spectrum of the tungsten cofactor. The steady-state data are consistent with a substrate substituted-enzyme mechanism for three substrates (formaldehyde plus two ferredoxin molecules). The KM value for free formaldehyde (21 microM) with ferredoxin as an electron acceptor is approximately 3 times lower than the value measured when benzyl viologen is used as an acceptor. The KM of ferredoxin (14 microM) is an order of magnitude less than previously reported values. An explanation for this discrepancy may be the fact that high concentrations of substrate are inhibitory and denaturing to the enzyme. Pre-steady-state difference spectra reveal peak shifts and a lack of isosbestic points, an indication that several processes happen in the first seconds of the reaction. Two fast processes (kobs1 = 4.7 s(-1), kobs2 = 1.9 s(-1)) are interpreted as oxidation of the substrate followed by rearrangement of the active site. Alternatively, these processes could be the entry/binding of the substrate followed by its oxidation. The release of the product and the electron shuffling over the tungsten and iron-sulfur center in the absence of an external electron acceptor are slower (kobs3 = 6.10 x 10(-2 )s(-1), kobs4 = 2.18 x 10(-2 )s(-1)). On the basis of these results in combination with results from previous electron paramagnetic resonance studies an activation route plus catalytic redox cycle is proposed.
| S-EPMC2099461 | biostudies-other
Project description:Inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the conversion of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP). The enzyme is an emerging target for antimicrobial therapy. The small molecule inhibitor A110 has been identified as a potent and selective inhibitor of IMPDHs from a variety of pathogenic microorganisms. A recent X-ray crystallographic study reported that the inhibitor binds to the NAD(+) cofactor site and forms a ternary complex with IMP. Here we report a pre-steady-state stopped-flow kinetic investigation of IMPDH from Bacillus anthracis designed to assess the kinetic significance of the crystallographic results. Stopped-flow kinetic experiments defined nine microscopic rate constants and two equilibrium constants that characterize both the catalytic cycle and details of the inhibition mechanism. In combination with steady-state initial rate studies, the results show that the inhibitor binds with high affinity (Kd ≈ 50 nM) predominantly to the covalent intermediate on the reaction pathway. Only a weak binding interaction (Kd ≈ 1 μM) is observed between the inhibitor and E·IMP. Thus, the E·IMP·A110 ternary complex, observed by X-ray crystallography, is largely kinetically irrelevant.
| S-EPMC5524190 | biostudies-literature
Project description:Mycobacterium tuberculosis and many other members of the Actinomycetes family produce mycothiol, i.e., 1-d-myo-inosityl-2-(N-acetyl-l-cysteinyl)amido-2-deoxy-alpha-d-glucopyranoside (MSH or AcCys-GlcN-Ins), to act against oxidative and antibiotic stress. The biosynthesis of MSH is essential for cell growth and has been proposed to proceed via a biosynthetic pathway involving four key enzymes, MshA-MshD. The MSH biosynthetic enzymes present potential targets for inhibitor design. With this as a long-term goal, we have carried out a kinetic and mechanistic characterization, using steady-state and pre-steady-state approaches, of the recombinant Mycobacterium smegmatis MshC. MshC catalyzes the ATP-dependent condensation of GlcN-Ins and cysteine to form Cys-GlcN-Ins. Initial velocity and inhibition studies show that the steady-state kinetic mechanism of MshC is a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding followed by cysteine binding, release of PPi, binding of GlcN-Ins, followed by the release of Cys-GlcN-Ins and AMP. The steady-state kinetic parameters were determined to be kcat equal to 3.15 s-1, and Km values of 1.8, 0.1, and 0.16 mM for ATP, cysteine, and GlcN-Ins, respectively. A stable bisubstrate analogue, 5'-O-[N-(l-cysteinyl)sulfamonyl]adenosine, exhibits competitive inhibition versus ATP and noncompetitive inhibition versus cysteine, with an inhibition constant of approximately 306 nM versus ATP. Single-turnover reactions of the first and second half reactions were determined using rapid-quench techniques, giving rates of approximately 9.4 and approximately 5.2 s-1, respectively, consistent with the cysteinyl adenylate being a kinetically competent intermediate in the reaction by MshC.
| S-EPMC2526253 | biostudies-literature
Project description:Plasma membrane glutamate transporters move glutamate across the cell membrane in a process that is thought to involve elevator-like movement of the transport domain relative to the static trimerization domain. Conformational changes associated with this elevator-like movement have been blocked by covalent crosslinking of cysteine pairs inserted strategically in several positions in the transporter structure, resulting in inhibition of steady-state transport activity. However, it is not known how these crosslinking restraints affect other partial reactions of the transporter that were identified based on pre-steady-state kinetic analysis. Here, we re-examine two different introduced cysteine pairs in the rat glutamate transporter EAAC1 recombinantely expressed in HEK293 cells, W440C/K268C and K64C/V419C, with respect to the molecular mechanism of their impairment of transporter function. Pre-steady-state kinetic studies of glutamate-induced partial reactions were performed using laser photolysis of caged glutamate to achieve sub-millisecond time resolution. Crosslinking of both cysteine pairs abolished steady-state transport current, as well as the majority of pre-steady-state glutamate-induced charge movements, in both forward and reverse transport mode, suggesting that it is not only the elevator-like movement associated with translocation, but also other transporter partial reactions that are inhibited. In contrast, sodium binding to the empty transporter, and glutamate-induced anion conductance were still intact after the W440C/K268C crosslink. Our results add to the previous mechanistic view of how covalent restraints of the transporter affect function and structural changes linked to individual steps in the transport cycle.
| S-EPMC9512041 | biostudies-literature
Project description:We used RNA-seq to profile E. coli K-12 MG1655 strains subjected to adaptive laboratory evolution after chorismate synthase knockouts. Either isochorismate synthase (menF) or isochorismate synthase AND chorismate lyase (ubiC) was deleted from a strain of E. coli K-12 MG1655 that had already been previously adapted for growth on glucose minimal media. RNA-seq profiles of the original glucose-adapted strain, the 2 deletion strains, and 4 laboratory-evolved strains from each deletion are included in duplicate. ubiC catalyzes the first committed step of ubiquinone synthesis, an important molecule for the electron transport chain. Thus, these experiments allowed assessment of cellular adaptations to restore energy metabolism capability.
2023-02-08 | GSE224889 | GEO
Project description:A comprehensive model for the mechanism of nitrogenase action is used to simulate pre-steady-state kinetic data for H2 evolution in the presence and in the absence of N2, obtained by using a rapid-quench technique with nitrogenase from Klebsiella pneumoniae. These simulations use independently determined rate constants that define the model in terms of the following partial reactions: component protein association and dissociation, electron transfer from Fe protein to MoFe protein coupled to the hydrolysis of MgATP, reduction of oxidized Fe protein by Na2S2O4, reversible N2 binding by H2 displacement and H2 evolution. Two rate-limiting dissociations of oxidized Fe protein from reduced MoFe protein precede H2 evolution, which occurs from the free MoFe protein. Thus Fe protein suppresses H2 evolution by binding to the MoFe protein. This is a necessary condition for efficient N2 binding to reduced MoFe protein.
| S-EPMC1144524 | biostudies-other