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Kinetic co-operativity of wheat-germ RNA polymerase II with adenosine 5'-[beta gamma-imido]triphosphate as substrate.

ABSTRACT: A kinetic study of productive RNA chain elongation indicates that adenosine 5'-[beta gamma-imido]triphosphate can serve as substrate in reactions catalysed by purified wheat-germ RNA polymerase II on a poly[d(A-T)] template. However, in contrast with the results obtained with the natural substrate ATP, the double-reciprocal plots, 1/velocity versus 1/[nucleotide], are not linear but characteristic of apparent negative co-operativity. The extent of the kinetic co-operativity is modified when the reactions are conducted in the additional presence of fixed amounts of an alternative substrate such as ATP or inhibitors such as dATP or cordycepin triphosphate. Analogous results are obtained whether the reactions are carried out in the presence of Mg2+ or Mn2+ as the metal ion cofactor. However, the data show that with Mn2+ the RNA polymerase is less specific in substrate recognition than with Mg2+. Tentative kinetic models are proposed to account for the rate measurements.

SUBMITTER: Job C

PROVIDER: S-EPMC1149106 | biostudies-other | 1988 May

REPOSITORIES: biostudies-other

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A slow kinetic transient in RNA synthesis catalysed by wheat-germ RNA polymerase II.

Project description:Progress curves of U-A-primed RNA synthesis catalysed by wheat-germ RNA polymerase II on a poly[d(A-T)] template exhibit a slow burst of activity. In contrast, the progress curves of single-step addition of UMP to U-A primer in the abortive elongation reaction do not exhibit the slow burst of activity. The correlation between the kinetic transient in the productive pathway of RNA synthesis and the rate of abortive elongation is suggestive of the occurrence of a slow conformational change of the transcription complex during the transition from abortive to productive elongation. The exceptional duration of the transient burst (in the region of 4 min) may suggest a transition of a hysteretic type.

| S-EPMC1149287 | biostudies-other

A calcium ion-dependent adenosine triphosphate pyrophosphohydrolase in plasma membrane from rat liver. Demonstration that the adenosine triphosphate analogues adenosine 5'-[betagamma-imido]triphosphate and adenosine 5'-[betagamma-methylene]-triphosphate are substrates for the enzyme.

Project description:An ATP pyrophosphohydrolase in a rat liver plasma-membrane subfraction was studied with respect to specific Ca2+ activation of the beta-phosphate bond hydrolysis. ATP and, in addition, adenosine 5'-[betagamma-imido]triphosphate and adenosine 5'-[betagamma-methlylene]triphosphate were substrates for Ca2+-stimulated enzymic hydrolysis of the beta-phosphate bond. A 15-fold activation was observed by raising the free Ca2+ concentration from 10(-7) to 10(-5) M. Mg2+ had little effect. Solubilization in 1% deoxycholate and partial purification on a sucrose density gradient resulted in a 5-fold increase in specific activity with unaltered Ca2+-stimulation pattern. The possible importance of the enzyme in Ca2+ transport is discussed.

| S-EPMC1184032 | biostudies-other

Kinetic Effects of β,γ-Modified Deoxynucleoside 5'-Triphosphate Analogues on RNA-Catalyzed Polymerization of DNA.

Project description:A recently described DNA polymerase ribozyme, obtained by in vitro evolution, provides the opportunity to investigate mechanistic features of RNA catalysis using methods that previously had only been applied to DNA polymerase proteins. Insight can be gained into the transition state of the DNA polymerization reaction by studying the behavior of various β,γ-bridging substituted methylene (CXY; X, Y = H, halo, methyl) or imido (NH) dNTP analogues that differ with regard to the pKa4 of the bisphosphonate or imidodiphosphate leaving group. The apparent rate constant (kpol) of the polymerase ribozyme was determined for analogues of dGTP and dCTP that span a broad range of acidities for the leaving group, ranging from 7.8 for the CF2-bisphosphonate to 11.6 for the CHCH3-bisphosphonate. A Brønsted plot of log(kpol) versus pKa4 of the leaving group demonstrates linear free energy relationships (LFERs) for dihalo-, monohalo-, and non-halogen-substituted analogues of the dNTPs, with negative slopes, as has been observed for DNA polymerase proteins. The unsubstituted dNTPs have a faster catalytic rate than would be predicted from consideration of the linear free energy relationship alone, presumably due to a relatively more favorable interaction of the β,γ-bridging oxygen within the active site. Although the DNA polymerase ribozyme is considerably slower than DNA polymerase proteins, it exhibits a similar LFER fingerprint, suggesting mechanistic commonality pertaining to the buildup of negative charge in the transition state, despite the very different chemical compositions of the two catalysts.

| S-EPMC8024617 | biostudies-literature

Purification and subunit analysis of wheat-germ ribonucleic acid polymerase II.

Project description:A procedure is described for the purification of the alpha-amanitin-sensitive DNA-dependent RNA polymerase [EC 2.7.7.6] from wheat germ. Solubilization of the enzyme activity was achieved by sonication of a crude extract in a high-salt buffer. Purification involved precipitation with protamine sulphate and (NH(4))(2)SO(4), chromatography on DEAE-cellulose and phosphocellulose, and sucrose gradient centrifugation. Under denaturing conditions the enzyme dissociated into five polypeptides with molecular weights and molar ratios of 220000 (0.9), 170000 (0.1), 140000 (1.0), 45000 (0.2), and 40000 (0.4). Approx. 1mg of purified RNA polymerase was obtained as a routine from 100g of starting material.

| S-EPMC1166341 | biostudies-other

Pre-steady-state kinetic studies establish entecavir 5'-triphosphate as a substrate for HIV-1 reverse transcriptase.

Project description:The novel 2'-deoxyguanosine analog Entecavir (ETV) is a potent inhibitor of hepatitis B virus (HBV) replication and is recommended for treatment in human immunodeficiency virus type 1 (HIV-1) and HBV-co-infected patients because it had been reported that ETV is HBV-specific. Recent clinical observations, however, have suggested that ETV may indeed demonstrate anti-HIV-1 activity. To investigate this question at a molecular level, kinetic studies were used to examine the interaction of 5'-triphosphate form of ETV with wild type (WT) HIV-1 reverse transcriptase (RT) and the nucleoside reverse transcriptase inhibitor-resistant mutation M184V. Using single turnover kinetic assays, we found that HIV-1 WT RT and M184V RT could use the activated ETV triphosphate metabolite as a substrate for incorporation. The mutant displayed a slower incorporation rate, a lower binding affinity, and a lower incorporation efficiency with the 5'-triphosphate form of ETV compared with WT RT, suggesting a kinetic basis for resistance. Our results are supported by cell-based assays in primary human lymphocytes that show inhibition of WT HIV-1 replication by ETV and decreased susceptibility of the HIV-1 containing the M184V mutation. This study has important therapeutic implications as it establishes ETV as an inhibitor for HIV-1 RT and illustrates the mechanism of resistance by the M184V mutant.

| S-EPMC7748947 | biostudies-literature

Transition State Analysis of Adenosine Triphosphate Phosphoribosyltransferase.

Project description:Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first step in histidine biosynthesis, a pathway essential to microorganisms and a validated target for antimicrobial drug design. The ATP-PRT enzyme catalyzes the reversible substitution reaction between phosphoribosyl pyrophosphate and ATP. The enzyme exists in two structurally distinct forms, a short- and a long-form enzyme. These forms share a catalytic core dimer but bear completely different allosteric domains and thus distinct quaternary assemblies. Understanding enzymatic transition states can provide essential information on the reaction mechanisms and insight into how differences in domain structure influence the reaction chemistry, as well as providing a template for inhibitor design. In this study, the transition state structures for ATP-PRT enzymes from Campylobacter jejuni and Mycobacterium tuberculosis (long-form enzymes) and from Lactococcus lactis (short-form) were determined and compared. Intrinsic kinetic isotope effects (KIEs) were obtained at reaction sensitive positions for the reverse reaction using phosphonoacetic acid, an alternative substrate to the natural substrate pyrophosphate. The experimental KIEs demonstrated mechanistic similarities between the three enzymes and provided experimental boundaries for quantum chemical calculations to characterize the transition states. Predicted transition state structures support a dissociative reaction mechanism with a DN*AN‡ transition state. Weak interactions from the incoming nucleophile and a fully dissociated ATP adenine are predicted regardless of the difference in overall structure and quaternary assembly. These studies establish that despite significant differences in the quaternary assembly and regulatory machinery between ATP-PRT enzymes from different sources, the reaction chemistry and catalytic mechanism are conserved.

| S-EPMC6693643 | biostudies-literature

Wheat-germ aspartate transcarbamoylase. Kinetic behaviour suggesting an allosteric mechanism of regulation.

Project description:1. Some kinetic properties of aspartate transcarbamoylase (EC 2.1.3.2), that had been purified approx. 20-fold from wheat germ, were studied. 2. A plot of enzyme activity against pH showed a low maximum at pH8.4 and a second, higher, maximum at pH10.5. A plot of percentage inhibition by 0.2mm-UMP against pH was approximately parallel to the plot of activity against pH, except that between pH6.5 and 7.5 the enzyme was insensitive to 0.2mm-UMP. 3. Kinetics were studied in detail at pH10.0 and 25 degrees C. In the absence of UMP, initial-rate plots were hyperbolic when the concentration of either substrate was varied. UMP decreased both V(max.) and K(m) in plots of initial rate against l-aspartate concentration, but the plots remained hyperbolic. However, UMP converted plots of initial rate against carbamoyl phosphate concentration into a sigmoidal shape, without significantly affecting V(max.). Plots of initial rate against UMP concentration were also sigmoidal. 4. The theoretical model proposed by Monod et al. (1965) gave a partial explanation of these results. When quasi-equilibrium conditions were assumed analysis in terms of this model suggested a trimeric enzyme binding the allosteric ligands, carbamoyl phosphate and UMP, nearly exclusively to the R and T conformational states respectively, and existing predominantly in the R state when ligands were absent. However, the values of the Hill coefficients for the co-operativity of each allosteric ligand were somewhat less than those predicted by the theory. 5. Some of the implications of these results are discussed, and the enzyme is contrasted with the well-known aspartate transcarbamoylase of Escherichia coli.

| S-EPMC1173766 | biostudies-other

Structural and kinetic analysis of nucleoside triphosphate incorporation opposite an abasic site by human translesion DNA polymerase η.

Project description:The most common lesion in DNA is an abasic site resulting from glycolytic cleavage of a base. In a number of cellular studies, abasic sites preferentially code for dATP insertion (the "A rule"). In some cases frameshifts are also common. X-ray structures with abasic sites in oligonucleotides have been reported for several microbial and human DNA polymerases (pols), e.g. Dpo4, RB69, KlenTaq, yeast pol ι, human (h) pol ι, and human pol β. We reported previously that hpol η is a major pol involved in abasic site bypass (Choi, J.-Y., Lim, S., Kim, E. J., Jo, A., and Guengerich, F. P. (2010 J. Mol. Biol. 404, 34-44). hpol η inserted all four dNTPs in steady-state and pre-steady-state assays, preferentially inserting A and G. In LC-MS analysis of primer-template pairs, A and G were inserted but little C or T was inserted. Frameshifts were observed when an appropriate pyrimidine was positioned 5' to the abasic site in the template. In x-ray structures of hpol η with a non-hydrolyzable analog of dATP or dGTP opposite an abasic site, H-bonding was observed between the phosphate 5' to the abasic site and water H-bonded to N1 and N6 of A and N1 and O6 of G nucleoside triphosphate analogs, offering an explanation for what appears to be a "purine rule." A structure was also obtained for an A inserted and bonded in the primer opposite the abasic site, but it did not pair with a 5' T in the template. We conclude that hpol η, a major copying enzyme with abasic sites, follows a purine rule, which can also lead to frameshifts. The phenomenon can be explained with H-bonds.

| S-EPMC4375460 | biostudies-literature

Adenosine triphosphate stimulates Aquifex aeolicus MutL endonuclease activity.

Project description:BACKGROUND:Human PMS2 (hPMS2) homologues act to nick 5' and 3' to misincorporated nucleotides during mismatch repair in organisms that lack MutH. Mn(++) was previously found to stimulate the endonuclease activity of these homologues. ATP was required for the nicking activity of hPMS2 and yPMS1, but was reported to inhibit bacterial MutL proteins from Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X)(2)E(X)(4)E motif present in the C-terminus of PMS2 homologues as important for endonuclease activity. METHODOLOGIES/PRINCIPAL FINDINGS:We examined the effect ATP had on the Mn(++) induced nicking of supercoiled pBR322 by full-length and mutant A. aeolicus MutL (Aae MutL) proteins. Assays were single time point, enzyme titration experiments or reaction time courses. The maximum velocity for MutL nicking was determined to be 1.6+/-0.08x10(-5) s(-1) and 4.2+/-0.3x10(-5) s(-1) in the absence and presence of ATP, respectively. AMPPNP stimulated the nicking activity to a similar extent as ATP. A truncated Aae MutL protein composed of only the C-terminal 123 amino acid residues was found to nick supercoiled DNA. Furthermore, mutations in the conserved C-terminal DQHA(X)(2)E(X)(4)E and CPHGRP motifs were shown to abolish Aae MutL endonuclease activity. CONCLUSIONS:ATP stimulated the Mn(++) induced endonuclease activity of Aae MutL. Experiments utilizing AMPPNP implied that the stimulation did not require ATP hydrolysis. A mutation in the DQHA(X)(2)E(X)(4)E motif of Aae MutL further supported the role of this region in endonclease activity. For the first time, to our knowledge, we demonstrate that changing the histidine residue in the conserved CPHGRP motif abolishes endonucleolytic activity of a hPMS2 homologue. Finally, the C-terminal 123 amino acid residues of Aae MutL were sufficient to display Mn(++) induced nicking activity.

| S-EPMC2744016 | biostudies-literature

The stereochemical course of yeast hexokinase-catalysed phosphoryl transfer by using adenosine 5'[gamma(S)-16O,17O,18O]triphosphate as substrate.

Project description:Adenosine 5'[gamma(S)-16O, 17O, 18O]triphosphate has been synthesized and used to determine the stereochemical course of phosphoryl transfer catalysed by yeast hexokinase. The chirality at phosphorus of the D-glucose 6-[16O,17O,18O]phosphate formed was analysed, after cyclization and methylation, by 31P n.m.r. spectroscopy. The phosphoryl transfer was found to occur with inversion of configuration, with a stereoselectivity in excess of 94%. The simplest interpretation of this result is that the phosphoryl group is transferred between substrates in the enzyme-substrate ternary complex by an 'in line' mechanism.

| S-EPMC1163354 | biostudies-other

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