A tetrahedral intermediate in the EPSP synthase reaction observed by rapid quench kinetics

Anderson, Karen S.; Sikorski, James A.; Johnson, Kenneth A.

doi:10.1021/bi00419a034

Cited by 160 publications

(197 citation statements)

References 21 publications

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“…Butylthiophosphate is very stable in the pH range 10 -14, whereas the thiophospho-PTS proteins are not. Above pH 12, the behavior of the two thiophospho-PTS proteins (19,20), or by nonlinear least squares fitting of the data from groups of experiments using Fitsim (21). The analyses were performed by first choosing a KЈ D for the sugar binding reaction; the table shows the results obtained for K D ϭ 10…”

Section: Resultsmentioning

confidence: 99%

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Transient State Kinetics of Enzyme IICBGlc, a Glucose Transporter of the Phosphoenolpyruvate Phosphotransferase System of Escherichia coli

Meadow

Savtchenko

Nezami³

et al. 2005

Journal of Biological Chemistry

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During translocation across the cytoplasmic membrane of Escherichia coli, glucose is phosphorylated by phospho-IIA Glc and Enzyme IICB Glc , the last two proteins in the phosphotransfer sequence of the phosphoenolpyruvate:glucose phosphotransferase system. Transient state (rapid quench) methods were used to determine the second order rate constants that describe the phosphotransfer reactions (phospho-IIA Glc to IICB Glc to Glc) and also the second order rate constants for the transfer from phospho-IIA to the phosphorylation site Cys of IIB Glc or IICB Glc were found to be 3.5 and 12, respectively. These equilibrium constants signify that the thiophospho-group in these proteins has a high phosphotransfer potential, similar to that of the phosphohistidinyl phosphotransferase system proteins. In these studies, preparations of IICB Glc were invariably found to contain endogenous, firmly bound Glc (estimated K D ϳ10 ؊7 M). The bound Glc was kinetically competent and was rapidly phosphorylated, indicating that IICB Glc has a random order, Bi Bi, substituted enzyme mechanism. The equilibrium constant for the binding of Glc was deduced from differences in the statistical goodness of fit of the phosphotransfer data to the kinetic model.The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) 3 comprises dozens of cytoplasmic and membrane proteins, most of which are the sugar-specific components of the system (for reviews, see Refs. 1-4). The PTS has several important functions in eubacterial cell physiology in addition to its major role in sugar transport, especially in Gram-positive bacteria (5).When the PTS catalyzes sugar transport, the sugar is phosphorylated as it crosses the membrane, and this process requires 3-6 proteins, depending on the sugar. One example is the Glc transport system in enteric bacteria, shown in Fig. 1.The first two proteins, Enzyme I and HPr, are the so-called general proteins of the system, meaning that they are not sugar-specific. The second pair of proteins, IIA Glc and IICB Glc , are the sugar-specific components. In other cases, the sugar-specific proteins vary from one to four separately encoded proteins. All of the phosphotransfer reactions except the last step (transfer to the sugar) are physiologically reversible. A second point is that the phosphorylation site residues in the PTS proteins are generally His, but in a number of the membrane proteins, the phosphorylation site can be a Cys residue. The phosphoryl group is transferred from PEP through a chain of His proteins to the membrane Enzyme II, where the phosphorylation site can be a His or a Cys (as it is in IICB Glc ), and finally to the sugar. In IICB Glc , the B domain extends into the cytoplasm and contains the phosphorylation site Cys.Our long term goal is to be able to predict the kinetics of Glc uptake by intact cells, and for this purpose it is necessary to obtain the rate constants for each of the reactions in Fig. 1. We established the basic methodology by developing a rapid quench method for determini...

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Section: Resultsmentioning

confidence: 99%

“…2 (20), was used manually to fit the mathematical models to the experimental data. When experimental data met the criteria for nonlinear least squares fitting (21), the Fitsim module of Kinsim was used.…”

Section: Kinetic Properties Of Endogenous Glc In Membrane Preparations-mentioning

confidence: 99%

Transient State Kinetics of Enzyme IICBGlc, a Glucose Transporter of the Phosphoenolpyruvate Phosphotransferase System of Escherichia coli

Meadow

Savtchenko

Nezami³

et al. 2005

Journal of Biological Chemistry

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“…1) using the kinetics fitting program KinTekSim (27). These data were fit "manually"; that is, the data were simulated using many different combinations of rate constants until the best fits were obtained.…”

Section: Methodsmentioning

confidence: 99%

Kinetic Investigation of Escherichia coli RNA Polymerase Mutants That Influence Nucleotide Discrimination and Transcription Fidelity

Holmes¹,

Santangelo

Cunningham³

et al. 2006

Journal of Biological Chemistry

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Recent RNA polymerase (RNAP) structures led to a proposed three-step model of nucleoside triphosphate (NTP) binding, discrimination, and incorporation. NTPs are thought to enter through the secondary channel, bind to an E site, rotate into a pre-insertion (PS) site, and ultimately align in the catalytic (A) site. We characterized the kinetics of correct and incorrect incorporation for several Escherichia coli RNAPs with substitutions in the proposed NTP entry pore (secondary channel). Substitutions of the semi-conserved residue ␤Asp 675 , which is >10Å away from these sites, significantly reduce fidelity; however, substitutions of the totally conserved residues ␤Arg 678 and ␤Asp 814 do not significantly alter the correct or incorrect incorporation kinetics, even though the corresponding residues in RNAPII crystal structures appear to be interacting with the NTP phosphate groups and coordinating the second magnesium ion in the active site, respectively. Structural analysis suggests that the lower fidelity of the ␤Asp 675 mutants most likely results from reduction of the negative potential of a small pore between the E and PS sites and elimination of several structural interactions around the pore. We suggest a mechanism of nucleotide discrimination that is governed both by rotation of the NTP through this pore and subsequent rearrangement or closure of RNAP to align the NTP in the A site.High resolution structures of multisubunit RNA polymerases (RNAPs) 4 have offered insight into many of the regulatory stages and conformational changes associated with the transcription cycle (1-14). Multiple structures of both prokaryotic and eukaryotic RNAPs have revealed a funnel-shaped pore (termed the secondary channel) leading from the surface of the enzyme directly to the active site. This channel has been proposed to be the major, and perhaps only, pathway for entry of nucleoside triphosphates (NTPs) to the active site (1-11, 13, 15).Recent structures of the yeast RNAP II elongation complex revealed that binding of an incorrect NTP for synthesis was at a site (termed the E site) adjacent to the catalytic NTP binding site (termed the A site) with the position of the base in the E site inverted and pointing out into the secondary channel (6). Comparison of structures with the correct and incorrect nucleotides led to the proposal of a two-step model of NTP binding, in which an incoming NTP first binds to the E site and then rotates through a narrow negatively charged pore (13) into the A site where it pairs with the template base (6). An NTP bound in the E site was also suggested to inhibit backtracking (13). This two-step model, however, does not allow for direct base pairing between the incoming NTP and the template base until the incoming NTP is positioned in the active site. Cramer and co-workers (11) observed an NTP analog in a third site in which the incoming NTP was base-paired with the DNA template base but not positioned for catalysis and suggested that this site was a preinsertion (PS) site. They proposed a mecha...

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“…The reaction mechanisms of both MurA and AroA have been studied intensively with pre-steady state experiments (11,14,15), site directed mutagenesis (11), substrate analogues (16)(17)(18), x-ray crystallography (7,19,20), fluorescence titration (21) and NMR experiments (6,22). Their mechanisms are similar in several aspects.…”

Section: General Acid Base Catalysis and Tetrahedral Intermediatementioning

confidence: 99%

“…In sucrose phosphorylase (37 39), 14 C in fructose (the free sugar product) is incorporated into the sucrose in the absence of inorganic phosphate. This suggests an enzyme covalently linked adduct is involved in the reaction (Fig.…”

Section: Reverse Reactionmentioning

confidence: 99%

Phosphate Analogues as Probes of the Catalytic Mechanisms of MurA and AroA, Two Carboxyvinyl Transferases

Zhang

Berti

2006

Biochemistry

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The two carboxyvinyl transferases MurA and AroA are essential for bacterial survival, and are proven or potential antibiotic targets. The reactions they catalyze are chemically challenging, involving protonation of an ethylene group in the first step, and deprotonation of a methyl in the second step. In order to probe how the enzymes promote these reactions, the reverse reactions from enolpyruvyl compounds (EP-OR) plus phosphate to phosphoenolpyruvate (PEP) plus R-OH were investigated, and compared with EP-OR hydrolysis reactions catalyzed by phosphate analogues.

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A tetrahedral intermediate in the EPSP synthase reaction observed by rapid quench kinetics

Cited by 160 publications

References 21 publications

Transient State Kinetics of Enzyme IICBGlc, a Glucose Transporter of the Phosphoenolpyruvate Phosphotransferase System of Escherichia coli

Transient State Kinetics of Enzyme IICBGlc, a Glucose Transporter of the Phosphoenolpyruvate Phosphotransferase System of Escherichia coli

Kinetic Investigation of Escherichia coli RNA Polymerase Mutants That Influence Nucleotide Discrimination and Transcription Fidelity

Phosphate Analogues as Probes of the Catalytic Mechanisms of MurA and AroA, Two Carboxyvinyl Transferases

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