Analysis of progress curves by simulations generated by numerical integration

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...

Section: Resultsmentioning

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

“…The distribution of A43a (active) and A43p (paused) states was determined as a function of stall time, as shown above. The data were fit to simple kinetic models, using the program KinTekSim (43,44). We found, however, that kinetic models required an unexpected feature.…”

Section: Resultsmentioning

confidence: 99%

“…Kinetic Modeling-Kinetic models were fit to experimental data using the program KinTekSim (43,44). No ATP-or GTP-dependent steps were considered in these models, because elongation from C40 was found to be largely independent of the ATP concentration, within a large concentration range (10 -250 M ATP), and modeling ATP-dependent steps was not necessary.…”

Section: Methodsmentioning

confidence: 99%

Combinatorial Control of Human RNA Polymerase II (RNAP II) Pausing and Transcript Cleavage by Transcription Factor IIF, Hepatitis δ Antigen, and Stimulatory Factor II

Zhang

Yan

Burton

2003

When RNA polymerase II (RNAP II) is forced to stall, elongation complexes (ECs) are observed to leave the active pathway and enter a paused state. Initially, ECs equilibrate between active and paused conformations, but with stalls of a long duration, ECs backtrack and become sensitive to transcript cleavage, which is stimulated by the EC rescue factor stimulatory factor II (TFIIS/SII). In this work, the rates for equilibration between the active and pausing pathways were estimated in the absence of an elongation factor, in the presence of hepatitis ␦ antigen (HDAg), and in the presence of transcription factor IIF (TFIIF), with or without addition of SII. Rates of equilibration between the active and paused states are not very different in the presence or absence of elongation factors HDAg and TFIIF. SII facilitates escape from stalled ECs by stimulating RNAP II backtracking and transcript cleavage and by increasing rates into and out of the paused EC. TFIIF and SII cooperate to merge the pausing and active pathways, a combinatorial effect not observed with HDAg and SII. In the presence of HDAg and SII, pausing is observed without stimulation of transcript cleavage, indicating that the EC can pause without backtracking beyond the pretranslocated state.Our laboratory has applied transient state kinetic analysis (1-3) to probe the mechanism and regulation of elongation by human RNA polymerase II (RNAP II) 1 (4, 5). In the current work, we concentrate on control of the branch point between the active synthesis pathway and the pausing pathway. We measure the rate by which RNAP II leaves the active synthesis pathway to enter the pausing pathway after encountering a barrier to elongation caused by withholding the next substrate nucleoside triphosphate.It has been suggested that transcription factor IIF (TFIIF) stimulates RNAP II elongation by suppressing transcriptional pausing (6 -8). TFIIF is a general initiation and elongation factor for RNAP II, composed of RAP74 and RAP30 subunits (RAP for RNA polymerase II-associating protein). In vitro, TFIIF stimulates elongation about 5-10-fold, achieving rates on chromatin-free DNA templates that are similar to rates observed on chromatinized templates in vivo (6 -12). Based on transient state kinetic studies, our laboratory recently proposed a mechanism for RNAP II elongation stimulated by TFIIF and hepatitis ␦ antigen (HDAg) (5). We have also demonstrated the major defects of the RAP74 I176A mutant in general and transient state elongation assays, providing insight into the functions of TFIIF in the RNAP II mechanism (11). We find that TFIIF exerts a global effect on elongation. TFIIF helps commit elongation complexes (ECs) to the forward synthesis pathway. TFIIF stimulates the rate of chemistry and accelerates a slow step after chemistry in the normal processive transition between bonds, a step that includes translocation and pyrophosphate release (5, 11). We conjecture that TFIIF binds to an external surface of RNAP II, tightens the RNAP II clamp, which grasps the RNA-DNA...

“…In calculating the amplitudes of absorbance and fluorescence changes, due correction was made for the signal undetected in the dead time. Rate constants were fitted to the kinetic scheme (see Reaction 2) using the programs KINSIM (28) and FITSIM (29).…”

Section: Methodsmentioning

confidence: 99%

Stopped-flow Reaction Kinetics of Recombinant Components of Proton-translocating Transhydrogenase with Physiological Nucleotides

Venning

Peake²,

Quirk

et al. 2000

Transhydrogenase, which is found in the inner membranes of animal mitochondria and the cytoplasmic membranes of some bacteria, catalyzes the reaction shown below.A single proton is translocated across the membrane, from the p-aqueous phase (the "outside" of intact mitochondria and bacteria) to the n-aqueous phase (the "inside"), for each hydride equivalent transferred from NADH to NADP ϩ . Under most conditions, this is the in vivo direction; the reaction is driven by the proton electrochemical gradient (⌬p) resulting from the action of respiratory (or photosynthetic) electron transport. For recent reviews, see Refs. 1-3.The gross structure of transhydrogenases from different species is strikingly invariant. The enzyme has three components; dI and dIII, which bind NAD(H) and NADP(H), respectively, protrude from the membrane (on the cytoplasmic side in bacteria, and the matrix side in mitochondria), and dII spans the membrane. Crystal structures of the dIII components of human and bovine transhydrogenases were recently described (4 -6), and the solution structure of the Rhodospirillum rubrum equivalent was determined by NMR.1 The basic fold of dIII is similar to the classical, dinucleotide-binding domain of lactate dehydrogenase, but NADP ϩ is bound with an unusual, "reversed" orientation. The nicotinamide ring of the bound NADP ϩ is exposed on a ridge of dIII. A homology model of dI (8), based on the crystal structure of the sequence-related alanine dehydrogenase (9), suggests that the nicotinamide ring of NADH is located in a deep cleft. It was proposed that, in the complete transhydrogenase, the ridge of dIII inserts into the cleft of dI to bring the nicotinamide rings of the two nucleotides into apposition to effect direct hydride transfer (5, 6). The protruding helix-D/loop-D of dIII is thought to interact with the membrane-spanning, dII and, together with the adjacent, lidlike loop E, which passes over the bound nucleotide, might be responsible for the energy transmission. The proton translocation steps during turnover are probably coupled specifically to changes in the mode of NADP(H) binding (1,5,10).Kinetic studies of transhydrogenase have also developed, following discoveries that fragments of the protein retain their nucleotide binding and some catalytic capacity. Thus, recombinant dI and dIII proteins, which bind their cognate nucleotides, have now been isolated from a number of species (11-16); mixtures of these proteins, even from different species, catalyze transhydrogenation reactions, albeit with properties that are modified relative to those of the complete enzyme. Transient state experiments, in particular, have revealed useful information on the hydride transfer step (17)(18)(19). Without exception, experiments with dI⅐dIII complexes have been carried out with nucleotide analogues having altered absorbance spectra to facilitate measurement of the rate of reaction. In this report we describe transient state experiments with the physiological nucleotides. A procedure is described, in which singl...