Analysis of numerical methods for computer simulation of kinetic processes: Development of KINSIM—A flexible, portable system

Barshop, Bruce A.; Wrenn, Richard F.; Frieden, Carl

doi:10.1016/0003-2697(83)90660-7

Cited by 703 publications

(646 citation statements)

References 13 publications

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“…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%

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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

Journal of Biological Chemistry

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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...

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Section: Resultsmentioning

confidence: 99%

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

Journal of Biological Chemistry

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“…The data generated from the catalytic trapping experiments were fit using the kinetic simulation program KINSIM (28).…”

Section: Methodsmentioning

confidence: 99%

Chemical Clamping Allows for Efficient Phosphorylation of the RNA Carrier Protein Npl3

Aubol

Ungs

Lukasiewicz³

et al. 2004

Journal of Biological Chemistry

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Protein kinases phosphorylate the appropriate protein substrate by recognizing residues both proximal and distal to the site of phosphorylation. Although these distal contacts may provide excellent binding affinities (low K m values) through stabilization of the enzymesubstrate complex, these contacts could reduce catalytic turnover (decrease k cat ) through slow phosphoprotein release. To investigate how protein kinases can overcome this problem and maintain both high substrate affinities and high turnover rates, the phosphorylation of the yeast RNA transport protein Npl3 by its natural protein kinase, Sky1p, was evaluated. Sky1p bound and phosphorylated Npl3 with a K m that was 2 orders of magnitude lower than a short peptide mimic representing the phosphorylation site and only proximal determinants. Surprisingly, this extraordinary difference is not the result of high affinity Npl3 binding. Rather, Npl3 achieves a low K m through a rapid and favorable phosphoryl transfer step. This step serves as a chemical clamp that locks the protein substrate in the active site without unduly stabilizing the product phosphoprotein and slowing its release. The chemical clamping mechanism offers an efficient means whereby a protein kinase can simultaneously achieve both high turnover and good substrate binding properties.The phosphorylation of hydroxyl-containing amino acids (i.e. serine, threonine, and tyrosine) in eukaryotic proteins controls numerous physiological responses essential for cell function. The enzymes that catalyze this modification, the protein kinases, have been studied for their critical role in complex signaling cascades and for their potential as valuable chemotherapeutic targets in proliferative diseases (1, 2). Although much is known about the protein kinase family on the structural and functional levels (3-6), less is known about the interactions of these enzymes with their physiological protein substrates. Protein kinases recognize and phosphorylate short peptide segments (consensus sequences) of 4 -10 residues based on the known phosphorylation sequences of their targets (7-9). Although these short segments (consensus sequences) are minimal substrates, they do not provide all the binding energy offered by the full-length protein substrate. For instance, the catalytic efficiency of phosphorylating the protein substrate MAPKAP2 using p38 MAPK 1 is ϳ2 orders of magnitude higher than that for a 14-residue peptide based on the consensus sequence (10). In another example, Csk phosphorylates the protein substrate Lck Ͼ3 orders of magnitude more efficiently than a 9-residue peptide based on the phosphorylation site (11). Studies of this type support a model in which regions beyond the limited consensus sequence interact with the protein kinase scaffold and govern high efficiency protein phosphorylation.Although an x-ray structure for a protein kinase with a full-length protein substrate bound is currently unavailable, structural and functional studies are now beginning to reveal the nature of the interactin...

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“…The program simulates chemical reactions by numerical integration of the underlying differential equations system. Integration intervals are determined by a flux-tolerance method [36]. It allows the simulation of combined rate equations and equilibria.…”

Section: Data Processingmentioning

confidence: 99%

Substrate specificity and stereoselectivity of horse liver alcohol dehydrogenase

Adolph

Maurer

Schneider-Bernlöhr

et al. 1991

European Journal of Biochemistry

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1. The steady-state parameters k,,, and K , and the rate constants of hydride transfer for the substrates isopropanol/acetone; (9-2-butano1, (R)-2-butanol/2-butanone; (9-2-pentano1, (R)-2-pentanol/2-pentanone; 3-pentanol/3-pentanone; (S)-2-octanol and (R)-2-octanol have been determined for the native Zn(I1)-containing horse-liver alcohol dehydrogenase (LADH) and the specific active-site-substituted Co(1I)LADH.2. A combined evaluation of steady-state kinetic data and rate constants obtained from stopped-flow measurements, allowed the determination of all rate constants of the following ordered bi-bi mechanism: E $ E . NAD 3. On the basis of the different substrate specificities of LADH and yeast alcohol dehydrogenase (YADH), a procedure has been developed to evaluate the enantiomeric product composition of ketone reductions. 2-Butanone and 2-pentanone reductions revealed (S)-Z-butanol(86%) and (S)-2-pentanol(95%) as the major products.4. The observed enantioselectivity implies the existence of two productive ternary complexes; E . NADH .(pro-S) 2-butdnone and E . NADH . (pro-R) 2-butanone. All rate constants describing the kinetic pathways of the system (S)-2-butanol, (R)-2-butanol/2-butanone have been determined. These data have been used to estimate the expected enantiomer product composition of 2-butanone reductions using apparent k,,,/K, values for the two different ternary-complex configurations of 2-butanone. Additionally, these data have been used for computer simulations of the corresponding reaction cycles. Calculated, simulated and experimental data were found to be in good agreement. Thus, the system (5')-2-butanol, (R)-2-butanol/2-butanone is the first example of a LADHcatalyzed reaction for which the stereochemical course could be described in terms of rate constants of the underlying mechanism. 5. The effects of Co(I1) substitution on the different steps of the kinetic pathway have been investigated. The free energy of activation is higher for alcohol oxidation and lower for ketone reduction when catalyzed by Co(1I)LADH in comparison to Zn(1I)LADH. However, the free energies of binding are affected by metal substitution in such a way that the enantioselectivity of ketone reduction is not significantly changed by the substitution of Co(I1) for Zn(I1).6. Evaluation of the data shows that substrate specificity and stereoselectivity result from combination of the free energies of binding and activation, with differences in binding energies as the dominating factors. In this regard, the interactions of substrate molecules with the protein moiety are dominant over the interactions with the catalytic metal ion.

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Analysis of numerical methods for computer simulation of kinetic processes: Development of KINSIM—A flexible, portable system

Cited by 703 publications

References 13 publications

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

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

Chemical Clamping Allows for Efficient Phosphorylation of the RNA Carrier Protein Npl3

Substrate specificity and stereoselectivity of horse liver alcohol dehydrogenase

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