The Experts below are selected from a list of 111027 Experts worldwide ranked by ideXlab platform

Nicholas Schramek - One of the best experts on this subject based on the ideXlab platform.

  • temperature dependent presteady state Kinetics of lumazine synthase from the hyperthermophilic eubacterium aquifex aeolicus
    Journal of Biological Chemistry, 2003
    Co-Authors: Ilka Haase, Markus Fischer, Adelbert Bacher, Nicholas Schramek
    Abstract:

    6,7-Dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyzes the condensation of 5-amino-6ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Presteady state kinetic experiments using the enzyme from the hyperthermophilic bacterium Aquifex aeolicus were monitored by multiwavelength photometry. An early optical transient absorbing around 330 nm is interpreted as a Schiff base intermediate obtained by reaction of the position 5 amino group of the heterocyclic substrate with the carbonyl group of 3,4-dihydroxy-2-butanone 4-phosphate. A second transient with an absorption maximum at 445 nm represents an intermediate resulting from the elimination of orthophosphate from the Schiff base. The rate-determining step is the subsequent formation of the 7-exomethylene type anion of 6,7-dimethyl-8ribityllumazine. The rate constants for the three partial reactions identified by the stopped flow experiments show linear Arrhenius relations in the temperature range of 15–70 °C.

Stephen W Ragsdale - One of the best experts on this subject based on the ideXlab platform.

  • mechanistic studies of methane biogenesis by methyl coenzyme m reductase evidence that coenzyme b participates in cleaving the c s bond of methyl coenzyme m
    Biochemistry, 2001
    Co-Authors: Yih-chern Horng, Donald F. Becker, Stephen W Ragsdale
    Abstract:

    Methyl-coenzyme M reductase (MCR), the key enzyme in methanogenesis, catalyzes methane formation from methyl-coenzyme M (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoBSH). Steady-state and Presteady-State Kinetics have been used to test two mechanistic models that contrast in the role of CoBSH in the MCR-catalyzed reaction. In class 1 mechanisms, CoBSH is integrally involved in methane formation and in C-S (methyl-SCoM) bond cleavage. On the other hand, in class 2 mechanisms, methane is formed in the absence of CoBSH, which functions to regenerate active MCR after methane is released. Steady-state kinetic studies are most consistent with a ternary complex mechanism in which CoBSH binds before methane is formed, as found earlier [Bonacker et al. (1993) Eur. J. Biochem. 217, 587-595]. Presteady-State kinetic experiments at high MCR concentrations are complicated by the presence of tightly bound CoBSH in the purified enzyme. Chemical quench studies in which (14)CH(3)-SCoM is rapidly reacted with active MCRred1 in the presence versus the absence of added CoBSH indicate that CoBSH is required for a single-turnover of methyl-SCoM to methane. Similar single turnover studies using a CoBSH analogue leads to the same conclusion. The results are consistent with class 1 mechanisms in which CoBSH is integrally involved in methane formation and in C-S (methyl-SCoM) bond cleavage and are inconsistent with class 2 mechanisms in which CoBSH binds after methane is formed. These are the first reported pre-steady-state kinetic studies of MCR.

Ilka Haase - One of the best experts on this subject based on the ideXlab platform.

  • temperature dependent presteady state Kinetics of lumazine synthase from the hyperthermophilic eubacterium aquifex aeolicus
    Journal of Biological Chemistry, 2003
    Co-Authors: Ilka Haase, Markus Fischer, Adelbert Bacher, Nicholas Schramek
    Abstract:

    6,7-Dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyzes the condensation of 5-amino-6ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Presteady state kinetic experiments using the enzyme from the hyperthermophilic bacterium Aquifex aeolicus were monitored by multiwavelength photometry. An early optical transient absorbing around 330 nm is interpreted as a Schiff base intermediate obtained by reaction of the position 5 amino group of the heterocyclic substrate with the carbonyl group of 3,4-dihydroxy-2-butanone 4-phosphate. A second transient with an absorption maximum at 445 nm represents an intermediate resulting from the elimination of orthophosphate from the Schiff base. The rate-determining step is the subsequent formation of the 7-exomethylene type anion of 6,7-dimethyl-8ribityllumazine. The rate constants for the three partial reactions identified by the stopped flow experiments show linear Arrhenius relations in the temperature range of 15–70 °C.

Yih-chern Horng - One of the best experts on this subject based on the ideXlab platform.

  • mechanistic studies of methane biogenesis by methyl coenzyme m reductase evidence that coenzyme b participates in cleaving the c s bond of methyl coenzyme m
    Biochemistry, 2001
    Co-Authors: Yih-chern Horng, Donald F. Becker, Stephen W Ragsdale
    Abstract:

    Methyl-coenzyme M reductase (MCR), the key enzyme in methanogenesis, catalyzes methane formation from methyl-coenzyme M (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoBSH). Steady-state and Presteady-State Kinetics have been used to test two mechanistic models that contrast in the role of CoBSH in the MCR-catalyzed reaction. In class 1 mechanisms, CoBSH is integrally involved in methane formation and in C-S (methyl-SCoM) bond cleavage. On the other hand, in class 2 mechanisms, methane is formed in the absence of CoBSH, which functions to regenerate active MCR after methane is released. Steady-state kinetic studies are most consistent with a ternary complex mechanism in which CoBSH binds before methane is formed, as found earlier [Bonacker et al. (1993) Eur. J. Biochem. 217, 587-595]. Presteady-State kinetic experiments at high MCR concentrations are complicated by the presence of tightly bound CoBSH in the purified enzyme. Chemical quench studies in which (14)CH(3)-SCoM is rapidly reacted with active MCRred1 in the presence versus the absence of added CoBSH indicate that CoBSH is required for a single-turnover of methyl-SCoM to methane. Similar single turnover studies using a CoBSH analogue leads to the same conclusion. The results are consistent with class 1 mechanisms in which CoBSH is integrally involved in methane formation and in C-S (methyl-SCoM) bond cleavage and are inconsistent with class 2 mechanisms in which CoBSH binds after methane is formed. These are the first reported pre-steady-state kinetic studies of MCR.

M P Lostao - One of the best experts on this subject based on the ideXlab platform.

  • effects of na and h on steady state and presteady state currents of the human concentrative nucleoside transporter 3 hcnt3
    Pflügers Archiv: European Journal of Physiology, 2010
    Co-Authors: Edurne Gorraitz, M P Lostao
    Abstract:

    Human concentrative nucleoside transporter 3 (hCNT3) uses the electrochemical gradient of Na+ and H+ to drive the transport of nucleosides and therapeutic nucleoside analogs into the cells. We employed the two-electrode voltage clamp technique to compare the steady-state and Presteady-State Kinetics of hCNT3 in the presence of Na+ and H+. We found that H+ supported a higher maximal rate of uridine transport than Na+, but the efficiency of transport was lower. For both cations, maximal uridine-induced current increased with hyperpolarizing potentials and did not saturate within the voltage range tested. Apparent affinity of hCNT3 for uridine in H+ was insensitive to membrane voltage at negative potentials, and decreased with depolarization. In contrast, apparent affinity for uridine in Na+ decreased with hyperpolarization and was independent of voltage at depolarizing potentials. H+-coupled hCNT3 exhibited lower affinity for all natural nucleosides and different substrate selectivity compared to Na+-coupled hCNT3. In H+, lack of the hydroxyl groups at 2′ and 5′ decreased the affinity, while lack of the nitrogen N-7 or inversion of the configuration of the hydroxyl group at 2′ prevented transport. Presteady-State charge movements of hCNT3 did not decrease when extracellular cation concentration (Na+ or H+) was reduced, but the τ ON–V and Q–V curves were shifted to more negative potentials. The different effects of uridine and inosine on Presteady-State currents in H+ indicated a change in rate-limiting step for the transport of these substrates by H+-coupled hCNT3.