scholarly journals Chorismate synthase. Pre-steady-state kinetics of phosphate release from 5-enolpyruvylshikimate 3-phosphate

1990 ◽  
Vol 265 (3) ◽  
pp. 899-902 ◽  
Author(s):  
T R Hawkes ◽  
T Lewis ◽  
J R Coggins ◽  
D M Mousdale ◽  
D J Lowe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Lag Phase ◽  
Chorismate Synthase ◽  
Phosphate Release ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Steady State Kinetics ◽  
Rate Limiting ◽  
Kinetics Of

The pre-steady-state kinetics of phosphate formation from 5-enolpyruvylshikimate 3-phosphate catalysed by Escherichia coli chorismate synthase (EC 4.6.1.4) were studied by a rapid-acid-quench technique at 25 degrees C at pH 7.5. No pre-steady-state ‘burst’ or ‘lag’ phase was observed, showing that phosphate is released concomitant with the rate-limiting step of the enzyme. The implications of this result for the mechanism of action of chorismate synthase are discussed.

scholarly journals The steady state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K12. The reduction of single-electron acceptors

1982 ◽  
Vol 203 (2) ◽  
pp. 505-510 ◽  
Author(s):  
R H Jackson ◽  
J A Cole ◽  
A Cornish-Bowden
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Nitrite Reductase ◽  
Maximum Velocity ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Steady State Kinetics ◽  
Cytochrome C Reduction ◽  
Pattern Of Variation ◽  
Rate Limiting

The kinetic characteristics of the diaphorase activities associated with the NADH-dependent nitrite reductase (EC 1.6.6.4) from Escherichia coli have been determined. The values of the apparent maximum velocity are similar for the reduction of Fe(CN)6(3)-and mammalian cytochrome c by NADH. These reactions may therefore have the same rate-limiting step. NAD+ activates NADH-dependent reduction of cytochrome c, and the apparent maximum velocity for this substrate increases more sharply with the concentration of NAD+ than for hydroxylamine. The simplest explanation is that NAD+ activation of hydroxylamine reduction derives solely from activation of steps involved in the reduction of cytochrome c, a flavin-mediated reaction, but these steps are only partly rate-limiting for the reduction of hydroxylamine. At 0.5 mM-NAD+, the apparent maximum velocity was 2.3 times higher for 0.1 mM-cytochrome c as substrate than for 100 mM-hydroxylamine, suggesting that the rate-limiting step during hydroxylamine reduction is a step that is not involved in cytochrome c reduction. A scheme is proposed that can account for the pattern of variation with [NAD+] of the Michaelis-Menten parameters for hydroxylamine and for NADH with hydroxylamine or cytochrome c as oxidized substrate.

scholarly journals A Novel Initiation Pathway in Escherichia Coli Transcription

2016 ◽  
Author(s):  
Eitan Lerner ◽  
SangYoon Chung ◽  
Benjamin L. Allen ◽  
Shuang Wang ◽  
Jookyung J. Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Magnetic Tweezer ◽  
Delayed Initiation ◽  
Rate Limiting ◽  
Kinetics Of

AbstractInitiation is a highly regulated, rate-limiting step in transcription. We employed a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with magnetic tweezer experiments and other methods, showed that, contrary to expectations, RNAP exit kinetics from later stages of initiation (e.g. from a 7-base transcript) was markedly slower than from earlier stages. Further examination implicated a previously unidentified intermediate in which RNAP adopted a long-lived backtracked state during initiation. In agreement, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states and prevented RNAP backtracking. Our results indicate a previously uncharacterized RNAP initiation state that could be exploited for therapeutic purposes and may reflect a conserved intermediate among paused, initiating eukaryotic enzymes.Significance:Transcription initiation by RNAP is rate limiting owing to many factors, including a newly discovered slow initiation pathway characterized by RNA backtracking and pausing. This backtracked and paused state occurs when all NTPs are present in equal amounts, but becomes more prevalent with NTP shortage, which mimics cellular stress conditions. Pausing and backtracking in initiation may play an important role in transcriptional regulation, and similar backtracked states may contribute to pausing among eukaryotic RNA polymerase II enzymes.

scholarly journals Some kinetic and steady-state properties of sodium channels after removal of inactivation.

1981 ◽  
Vol 77 (1) ◽  
pp. 1-22 ◽  
Author(s):  
G S Oxford
Keyword(s):  
Steady State ◽  
Activation Kinetics ◽  
Turn On ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Voltage Dependent ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Secondary Activation ◽  
Pronase Treatment

To study the kinetic and steady-state properties of voltage-dependent sodium conductance activation, squid giant axons were perfused internally with either pronase or N-bromoacetamide and voltage clamped. Parameters of activation, tau m and gNa(V), and deactivation, tau Na, were measured and compared with those obtained from control axons under the assumption that gNa oc m3h of the Hodgkin-Huxley scheme. tau m(V) values obtained from the turn-on of INa agree well with control axons and previous determinations by others. tau Na(V) values derived from Na tail currents were also unchanged by pronase treatment and matched fairly well previously published values. tau m(V) obtained from 3 x tau Na(V) were much larger than tau m(V) obtained from INa turn-on at the same potentials, resulting in a discontinuous distribution. Steady-state In (gNa/gNa max - gNa) vs. voltage was not linear and had a limiting logarithmic slope of 5.3 mV/e-fold gNa. Voltage step procedures that induce a second turn-on of INa during various stages of the deactivation (Na tail current) process reveal quasiexponential activation at early stages that becomes increasingly sigmoid as deactivation progresses. For moderate depolarizations, primary and secondary activation kinetics are superimposable. These data suggest that, although m3 can describe the shape of INa turn-on, it cannot quantitatively account for the kinetics of gNa after repolarization. Kinetic schemes for gNa in which substantial deactivation occurs by a unique pathway between conducting and resting states are shown to be unlikely. It appears that the rate-limiting step in linear kinetic models of activation may be between a terminal conducting state and the adjacent nonconducting intermediate.

Functional and structural roles of the glutathione-binding residues in maize (Zea mays) glutathione S-transferase I

2001 ◽  
Vol 358 (1) ◽  
pp. 101-110 ◽  
Author(s):  
Nikolaos E. LABROU ◽  
Luciane V. MELLO ◽  
Yannis D. CLONIS
Keyword(s):  
Steady State ◽  
Catalytic Mechanism ◽  
Limited Proteolysis ◽  
Glutathione S Transferase ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Steady State Kinetics ◽  
Helical Segment ◽  
Α Helix ◽  
Rate Limiting

The isoenzyme glutathione S-transferase (GST) I from maize (Zea mays) was cloned and expressed in Escherichia coli, and its catalytic mechanism was investigated by site-directed mutagenesis and dynamic studies. The results showed that the enzyme promotes proton dissociation from the GSH thiol and creates a thiolate anion with high nucleophilic reactivity by lowering the pKa of the thiol from 8.7 to 6.2. Steady-state kinetics fit well to a rapid equilibrium, random sequential Bi Bi mechanism, with intrasubunit modulation between the GSH binding site (G-site) and the electrophile binding site (H-site). The rate-limiting step of the reaction is viscosity-dependent, and thermodynamic data suggest that product release is rate-limiting. Five residues of GST I (Ser11, His40, Lys41, Gln53 and Ser67), which are located in the G-site, were individually replaced with alanine and their structural and functional roles in the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction were investigated. On the basis of steady-state kinetics, difference spectroscopy and limited proteolysis studies it is concluded that these residues: (1) contribute to the affinity of the G-site for GSH, as they are involved in side-chain interaction with GSH; (2) influence GSH thiol ionization, and thus its reactivity; (3) participate in kcat regulation by affecting the rate-limiting step of the reaction; and (4) in the cases of His40, Lys41 and Gln53 play an important role in the structural integrity of, and probably in the flexibility of, the highly mobile short 310-helical segment of α-helix 2 (residues 35–46), as shown by limited proteolysis experiments. These structural perturbations are probably transmitted to the H-site through changes in Phe35 conformation. This accounts for the modulation of Kcdnbm by His40, Lys41 and Gln53, and also for the intrasubunit communication between the G- and H-sites. Computer simulations using CONCOORD were applied to maize GST I monomer and dimer structures, each with bound lactoylglutathione, and the results were analysed by the essential dynamics technique. Differences in dynamics were found between the monomer and the dimer simulations showing the importance of using the whole structure in dynamic analysis. The results obtained confirm that the short 310-helical segment of α-helix 2 (residues 35–46) undergoes the most significant structural rearrangements. These rearrangements are discussed in terms of enzyme catalytic mechanism.

Kinetics of cyclization of S-ethoxycarbonylmethylisothiouronium chloride to 2-imino-4-thiazolidone

1979 ◽  
Vol 44 (3) ◽  
pp. 912-917 ◽  
Author(s):  
Vladimír Macháček ◽  
Said A. El-bahai ◽  
Vojeslav Štěrba
Keyword(s):  
Hydrochloric Acid ◽  
Dilute Hydrochloric Acid ◽  
Base Catalysis ◽  
General Base ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Kinetics Of Formation ◽  
Acid Catalyzed ◽  
Rate Limiting ◽  
Kinetics Of

Kinetics of formation of 2-imino-4-thiazolidone from S-ethoxycarbonylmethylisothiouronium chloride has been studied in aqueous buffers and dilute hydrochloric acid. The reaction is subject to general base catalysis, the β value being 0.65. Its rate limiting step consists in acid-catalyzed splitting off of ethoxide ion from dipolar tetrahedral intermediate. At pH < 2 formation of this intermediate becomes rate-limiting; rate constant of its formation is 2 . 104 s-1.

Kinetics and mechanism of cyclization of N-(2-methoxycarbonylphenyl)-N’-methylsulphonamide to 3-methyl-(1H)-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide

1991 ◽  
Vol 56 (8) ◽  
pp. 1701-1710 ◽  
Author(s):  
Jaromír Kaválek ◽  
Vladimír Macháček ◽  
Miloš Sedlák ◽  
Vojeslav Štěrba
Keyword(s):  
Potassium Hydroxide ◽  
Acid Catalysis ◽  
Potassium Hydroxide Solution ◽  
Hydroxide Solution ◽  
General Base ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Cyclization Kinetics ◽  
Rate Limiting ◽  
Kinetics Of

The cyclization kinetics of N-(2-methylcarbonylphenyl)-N’-methylsulfonamide (IIb) into 3-methyl-(1H)-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide (Ib) has been studied in ethanolamine, morpholine, and butylamine buffers and in potassium hydroxide solution. The cyclization is subject to general base and general acid catalysis. The value of the Bronsted coefficient β is about 0.1, which indicates that splitting off of the proton from negatively charged tetrahedral intermediate represents the rate-limiting and thermodynamically favourable step. In the solutions of potassium hydroxide the cyclization of dianion of the starting ester IIb probably becomes the rate-limiting step.

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