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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thiourea dioxide was used in chemical modification studies to identify functionally important amino acids in Escherichia coli CTP synthetase. Incubation at pH 8.0 in the absence of substrates led to rapid, time dependent, and irreversible inactivation of the enzyme. The second-order rate constant for inactivation was 0.18 M-1 s-1. Inactivation also occurred in the absence of oxygen and in the presence of catalase, thereby ruling out mixed-function oxidation/reduction as the mode of amino acid modification. Saturating concentrations of the substrates ATP and UTP, and the allosteric activator GTP prevented inactivation by thiourea dioxide, whereas saturating concentrations of glutamine (a substrate) did not. The concentration dependence of nucleotide protection revealed cooperative behavior with respect to individual nucleotides and with respect to various combinations of nucleotides. Mixtures of nucleotides afforded greater protection against inactivation than single nucleotides alone, and a combination of the substrates ATP and UTP provided the most protection. The Hill coefficient for nucleotide protection was approximately 2 for ATP, UTP, and GTP. In the presence of 1:1 ratios of ATP:UTP, ATP:GTP, and UTP:GTP, the Hill coefficient was approximately 4 in each case. Fluorescence and circular dichroism measurements indicated that modification by thiourea dioxide causes detectable changes in the structure of the protein. Modification with [14C]thiourea dioxide demonstrated that complete inactivation correlates with incorporation of 3 mol of [14C]thiourea dioxide per mole of CTP synthetase monomer. The specificity of thiourea dioxide for lysine residues indicates that one or more lysines are most likely involved in CTP synthetase activity. The data further indicate that nucleotide binding prevents access to these functionally important residues.
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PMID:Inactivation and covalent modification of CTP synthetase by thiourea dioxide. 130 49

Uridine di- and triphosphopyridoxals were used to probe the substrate-binding site in potato tuber UDP-glucose pyrophosphorylase (EC 2.7.7.9). The enzyme was rapidly inactivated in time- and dose-dependent manners when incubated with either reagent followed by reduction with sodium borohydride. The inactivations were almost completely retarded by UDP-Glc and UTP but only slightly by alpha-D-glucose 1-phosphate. The complete inactivation corresponded to the incorporation of about 0.9-1.0 mol of either reagent per mole of enzyme monomer. Both reagents appear to bind specifically to the UDP-Glc-(UTP)-binding site. Structural studies of the labeled enzymes revealed that the two reagents modified the identical set of five lysyl residues (Lys-263, Lys-329, Lys-367, Lys-409, and Lys-410), in which Lys-367 was most prominently modified. The ratios of the amounts of labels incorporated into these residues were similar for the two reagents. Furthermore, linear relationships were observed between the residual activities and the amounts of incorporation into each lysyl residue. We conclude that the five lysyl residues are located at or near the UDP-Glc(UTP)-binding site of potato tuber UDP-Glc pyrophosphorylase and that the modification of these residues occurs in a mutually exclusive manner, leading to the inactivation of the enzyme.
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PMID:Identification of lysyl residues located at the substrate-binding site in UDP-glucose pyrophosphorylase from potato tuber: affinity labeling with uridine di- and triphosphopyridoxals. 190 67

The Ca(++) transport mechanism in the red cell membrane was studied in resealed ghost cells. It was found that the red cell membrane can transport Ca(++) from inside the cell into the medium against great concentration gradient ratios. Tracing the movement of (45)Ca infused inside red cells indicated that over 95% of all Ca(++) in the cells was transported into media in 20 min incubation under the optimum experimental conditions. The influence of temperature on the rate constant of transport indicated an activation energy of 13,500 cal per mole. The optimum pH range of media for the transport was between 7.5 and 8.5. As energy sources, ATP(1), CTP, and UTP were about equally effective, GTP somewhat less effective, and ITP least effective among the nucleotides tested. The Ca(++) transport does not appear to involve exchange of Ca(++) with any monovalent or divalent cations. Also, it is not influenced by oligomycin, sodium azide, or ouabain in high concentrations, which inhibit the Ca(++) transport in mitochondria or in sarcoplasmic reticulum. In these respects, the Ca(++) transport mechanism in the red cell membrane is different from those of mitochondria and the sarcoplasmic reticulum.
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PMID:Studies on the active transport of calcium in human red cells. 535 89

In order to follow the kinetics of the initiation of transcription by the E. coli RNA polymerase, we have used the procedure of abortive initiation as described by Mc Clure (1980) (7). In place of radioactive labeling we have taken advantage of a fluorescent probe (UTP gamma ANS) to obtain fast and accurate determinations of the rate of transcription and to deduce from kinetic equations both the binding constant (KB) and the rate of isomerization (k2) which characterize the classical two-step model. This analysis was applied to the tet promoter of pBR322 in a linearized plasmid DNA and was studied in function of temperature (from 25 degrees C to 37 degrees C) and of pH (from 6 to 8.3). The association is entropy driven (delta H degrees = 29 Kcal/mole and delta S degrees = 130 e.u.). The activation energy of isomerization is 13 Kcal/mole. Both k2 and k-2 are increasing with pH. The insensitivity to pH of the KBK2 product could be tentatively explained in terms of the processive aspect of the polymerase binding to its specific site.
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PMID:A new experimental approach for studying the association between RNA polymerase and the tet promoter of pBR322. 636 39

The stoichiometry of the enzymatic reaction catalyzed by CTP synthetase from Escherichia coli was analyzed by high-performance liquid chromatography. The results revealed that for every mole of UTP transformed to CTP, one mole of ATP was converted to ADP. The substrate specificity of CTP synthetase from E. coli was investigated by means of UTP analogs. Chemical modification of UTP involved either the uracil, ribose or 5'-triphosphate part. None of the UTP analogs studied proved to be a substrate. The capacity of the UTP analogs to inhibit CTP synthetase was investigated. From the UTP derivatives employed only 2-thiouridine 5'-triphosphate was found to inhibit the enzyme competitively with reasonable affinity: Ki/Km(UTP) = 1. This study indicated that the three main structural elements of the UTP molecule: uracil, ribose and 5'-triphosphate moiety, contribute to substrate specificity. The behaviour of a limited number of CTP analogs as product-like inhibitors supported this view.
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PMID:Substrate specificity of CTP synthetase from Escherichia coli. 675 17

We have examined multiple cofactor usage by yeast tRNA ligase in splicing in vitro. The ligase mechanism of action requires expenditure of two molar equivalents of nucleotide cofactor per mole of tRNA product. Recent evidence (Westaway, S.K., Belford, H.G., Apostol, B.L., Abelson, J., and Greer, C.L. (1993) J. Biol. Chem. 268, 2435-2443) demonstrated that the ligase-associated kinase activity is more efficient with GTP as cofactor than with ATP. Employing a ligase fusion construct with dihydrofolate reductase (Apostol, B.L., Westaway, S.K., Abelson, J., and Greer, C.L. (1991) J. Biol. Chem. 266, 7445-7455) for purposes of enzyme purification, we performed joining assays demonstrating that ATP and GTP are the most effective combination of cofactors. ATP was essential to the joining reaction, while UTP, CTP, or ATP replaced GTP inefficiently. Specific and functionally independent binding sites were confirmed for ATP and GTP by direct binding measurement. A third site was implicated in UTP- and CTP-ligase interactions. Comparison of binding constants with Kapp values determined for nucleotide-dependent joining suggested both that nucleotide triphosphate binding may be limiting in tRNA joining and that tRNA ligation occurs most efficiently using GTP for the kinase reaction and ATP as the adenylylate synthetase cofactor.
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PMID:Multiple nucleotide cofactor use by yeast ligase in tRNA splicing. Evidence for independent ATP- and GTP-binding sites. 842 19

DNA-dependent RNA polymerases (RNApol) are Zn2+ metalloproteins where the Zn2+ ion plays both catalytic and structural roles. Although the ubiquitous presence of Zn2+ with the RNApol from eukaryotes had already been established, the exact stoichiometry of Zn2+ ion(s) per mole enzyme is not well documented, and its role in enzymatic function remains elusive. We show here that RNApolII from Saccharomyces cerevisiae has two Zn2+ ions tightly associated with it which are necessary for its transcriptional activity. Upon prolonged dialysis against 10 mM EDTA for 4-5 h, the enzyme loses one Zn2+, as well as partial activity. However, Zn2+ can be added back to the enzyme, but without recovering its total activity. 5 mM orthophenanthroline (OP) removes one Zn2+ within 2 h; the enzyme, however, cannot be reconstituted back with Zn2+. Circular dichroism (CD) studies showed that the conformation of the native enzyme is unique and cannot be reproduced with Zn2+-reconstituted RNApolII. Similarly, the rate of abortive synthesis of a dinucleotide product over a non-specific template is faster when catalyzed by two Zn2+-native enzymes. Zn2+-reconstituted RNApolII or one Zn2+-RNApolII showed a slower abortive synthesis rate. 65Zn2+-blotting experiments indicated that the removal of one Zn2+ from the enzyme destroys the Zn2+-binding ability of the larger subunits of yeast RNApolII. In order to check whether the presence of Zn2+ ions has any effect on substrate recognition, we followed the binding of (gamma-AmNS)UTP, a fluorescent substrate analog to RNApolII. It was observed that OP-treated enzyme showed non-specific substrate recognition, whereas two Zn2+-native RNApol binds substrate at a single site.
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PMID:The presence of two tightly bound Zn2+ ions is essential for the structural and functional integrity of yeast RNA polymerase II. 918 52

We have utilized DSC and high pressure FTIR spectroscopy to study the specificity and mechanism by which ATP protects actin against heat and pressure denaturation. Analysis of the thermograms shows that ATP raises the transition temperature Tm for actin from 69.6 to 75.8 degrees C, and the calorimetric enthalpy, deltaH, from 680 to 990 kJ/mole. Moreover, the peak becomes sharper indicating a more cooperative process. Among the other nucleotide triphosphates, only UTP increases the Tm by 2.5 degrees C, whereas GTP and CTP have negligable effects; ADP and AMP are less active, increasing the Tm by 2.1 and 1.6 degrees C, respectively. Therefore, gamma phosphate plays a key role in this protection, but its hydrolysis is not implicated since the nonhydrolysable analogue of ATP, ATP-PNP have the same activity as ATP. FTIR spectroscopy demonstrates that ATP also protects actin against high pressure denaturation. Analysis of the amide I band during the increase in pressure clearly illustrates that ATP protects particularly a region rich in beta-sheets of the actin molecule.
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PMID:Effects of nucleotides on the denaturation of F actin: a differential scanning calorimetry and FTIR spectroscopy study. 924 36

The 2',3'-dialdehyde derivative of ATP (dial-ATP) has been shown to be an affinity label for the ATP binding site of the H(+)-ATPase from tonoplast of etiolated mung bean seedlings (Vigna radiata L.). The dial-ATP caused marked inactivation of enzymatic activities of both membrane-bound and soluble ATPase and its associated proton translocation. The inactivation was reversible, but could be stabilized by NaBH(4). The sodium dodecyl sulfatepolyacrylamide gel electrophoresis pattern revealed that the dial-ATP binding site was in the large (A) subunit of ATPase. The inhibition could be substantially protected by its physiological substrate ATP, pyrophosphate, and nucleotides in the decreasing order: ATP > pyrophosphate > ADP = AMP > GTP > CTP = UTP. A Lineweaver-Burk plot showed that the mode of inhibition was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first order kinetics with a K(i) of 4.1 millimolar, a minimum inactivation half-time of 20 seconds, and a pseudo-first order rate constant of 0.035 s(-1). The double logarithmic plot of apparent rate constant versus dial-ATP concentration gave a slope of 0.927, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labeling studies with [(3)H]dial-ATP indicate that the incorporation of approximately 1 mole of dial-ATP per mole ATPase is sufficient to completely inhibit the ATPase. A working model of nonequivalent subunits for enzymatic mechanism of vacuolar ATPase is suggested.
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PMID:Inhibition of tonoplast ATPase by 2',3'-dialdehyde derivative of ATP. 1666 47