Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Salmonella typhimurium and Escherichia coli MutS protein is one of several methyl-directed mismatch repair proteins that act together to correct replication errors. MutS is homologous to the Streptococcus pneumoniae HexA mismatch repair protein and to the Duc1 and Rep1 proteins of human and mouse. Homology between the deduced amino acid sequence of both MutS and HexA, and the type A nucleotide binding site consensus sequence, suggested that ATP binding and hydrolysis play a role in their mismatch repair functions. We found that MutS does indeed weakly hydrolyze ATP to ADP and Pi, with a Km of 6 microM and kcat of 0.26. To show that this activity is intrinsic to MutS, we made a site-directed mutation, which resulted in the invariant lysine of the nucleotide binding consensus sequence being changed to an alanine. The mutant MutS allele was unable to complement a mutS::Tn10 mutation in vivo, and was dominant over wild type when present in high copy number. The purified mutant protein had reduced ATPase activity, with the Km affected more severely than the kcat. Like the wild type MutS protein, the mutant protein is able to bind heteroduplex DNA specifically, but the mutant protein does so with a reduced affinity.
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PMID:Altering the conserved nucleotide binding motif in the Salmonella typhimurium MutS mismatch repair protein affects both its ATPase and mismatch binding activities. 165 Dec 34

The insecticides mirex and chlordecone have previously been found to suppress the biliary excretion of a wide variety of compounds. In the present studies, the effects of mirex, chlordecone, and phenobarbital on the uptake of two endogenous organic anions, estradiol-17 beta(beta-D-glucuronide) (E217G), an estrogen metabolite, taurocholate (TC), a common bile acid, and an essential amino acid, L-alanine (L-Ala) (0.5 mM), into isolated rat hepatocytes was investigated. Female Sprague-Dawley rats were orally dosed with mirex (12.5, 25, and 50 mg/kg) or chlordecone (6.25, 12.5, and 18.75 mg/kg) dissolved in corn oil for 3 days and isolated rat hepatocytes were prepared 2 days later. Rats were also dosed orally with phenobarbital (50 mg/kg on the first day and 80 mg/kg for the next 4 days) dissolved in distilled deionized water, and isolated hepatocytes were prepared on the sixth day. Mirex significantly reduced the uptake of both organic anions (0.5, 10, and 50 microM E2 17G; 10 microM TC) into hepatocytes by 40-70%, whereas chlordecone had no effect on their uptake. Mirex at 50 mg/kg significantly reduced the Vmax for the low- and high-affinity E217G uptake sites by 70% and decreased the Km for the low affinity uptake site by 60%. Mirex also significantly decreased the Vmax for TC uptake from 1.11 to 0.82 nmol/min/mg protein but had no effect on its Km (23.2 vs 22.9 microM). Mirex at 50 mg/kg was also found to reduce the uptake of 0.5 mM L-Ala by nearly 40%. Phenobarbital had no effect on the uptake of E217G (0.5 microM), TC (10 microM), or L-Ala (0.5 mM). Mirex treatment had no effect on hepatic plasma membrane Na+,K(+)- or Mg2(+)-ATPase activity. Neither mirex nor chlordecone at 50-100 microM had any effect on the uptake of 10 microM TC when added directly to hepatocytes from naive rats. These results indicate that mirex decreases the transport of organic anions and L-Ala across the basolateral domain of the hepatocyte in addition to its inhibitory effects on biliary excretion.
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PMID:Mirex exposure inhibits the uptake of estradiol-17 beta(beta-D-glucuronide), taurocholate, and L-alanine into isolated rat hepatocytes. 169 54

Escherichia coli rho protein facilitates transcription termination by a mechanism that involves rho binding to the nascent RNA, activation of rho's RNA-dependent ATPase activity, and release of the mRNA from the DNA template. The initial step, formation of a rho-RNA complex, is mediated primarily by an RNA binding domain included within the amino-terminal 151 amino acids of rho protein. We have now identified one specific portion of this region that is involved in RNA binding, by photocross-linking and by site-directed mutagenesis. UV irradiation of rho-RNA complexes results in covalent attachment of the RNA to a single peptide in rho that apparently spans amino acids 45-100. Within this peptide is a ribonucleoprotein (RNP1) consensus sequence, Gly-Phe-Gly-Phe, that is present in many RNA-binding proteins. Mutagenesis of the phenylalanine residues in this consensus to leucine or alanine results in mutant proteins that are defective for RNA binding and have altered ATPase and RNA-DNA helicase activities. The weakened affinity but increased salt sensitivity of RNA binding by the mutant proteins suggests that they have lost more than just a set of nonionic interactions and are consistent with a change in the conformation of the RNA binding site. Whatever the changes, they appear localized primarily to the RNA binding domain because the mutants retain much of their RNA-dependent ATPase activity. We infer that the Phe residues themselves do not play a substantial role in the activation of ATP hydrolysis. Our results indicate that several different components of RNA interaction are required for rho activity and support a role for the RNP1 consensus region of rho in at least one specific aspect of RNA binding.
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PMID:Mutations in an RNP1 consensus sequence of Rho protein reduce RNA binding affinity but facilitate helicase turnover. 171 28

The effects of amino acid substitutions in the carboxyl terminal region of the H(+)-ATPase a subunit (271 amino acid residues) of Escherichia coli were studied using a defined expression system for uncB genes coded by recombinant plasmids. The a subunits with the mutations, Tyr-263----end, Trp-231----end, Glu-219----Gln, and Arg-210----Lys (or Gln) were fully defective in ATP-dependent proton translocation, and those with Gln-252----Glu (or Leu), His-245----Glu, Pro-230----Leu, and Glu-219----His were partially defective. On the other hand, the phenotypes of the Glu-269----end, Ser-265----Ala (or end), and Tyr-263----Phe mutants were essentially similar to that of the wild-type. These results suggested that seven amino acid residues between Ser-265 and the carboxyl terminus were not required for the functional proton pathway but that all the other residues except Arg-210, Glu-219, and His-245 were required for maintaining the correct conformation of the proton pathway. The results were consistent with a report that Arg-210 is directly involved in proton translocation.
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PMID:Role of the carboxyl terminal region of H(+)-ATPase (F0F1) a subunit from Escherichia coli. 182 13

Mutations have been made in the exposed region of the avian troponin C central helix, the D/E linker, which change its length and the orientation of the Ca2(+)-binding domains relative to each other. The region 87Glu-Asp-Ala-Lys-Gly-Lys-Ser-Glu-Glu-Glu97 has been altered in five deletion (d) mutants: dEDA, dKG, dKGK, dSEEE, and dKEDAKGK. The recombinant troponin Cs were expressed in Escherichia coli, purified, and assayed for function. All mutants retained basic troponin C function. They all bound Ca2+ to the low and high affinity sites, and they all were able to confer Ca2+ sensitivity on the regulated actomyosin ATPase. However, the regulatory function of all mutants except dSEEE was defective in one part of the Ca2+ switch or the other. In certain conditions dKGK and dKEDAKGK failed to inhibit fully whereas dEDA and dKG failed to activate the regulated actomyosin ATPase fully. The following general conclusions have been made. (a) The length of the D/E linker per se (assuming the linker is helical) and the orientation of the two Ca2(+)-binding domains relative to each other are not crucial for regulation. (b) The conserved charge cluster 95Glu-Glu-Glu97, in a region of troponin C known to bind to troponin I and postulated to be required for regulation, appears to be unimportant for function. (c) Deletion of 88Glu-Asp-Ala90 resulted in a troponin C that could not activate the actomyosin (or S1) ATPase over the level of actomyosin alone, thus defining a role for troponin C in this aspect of thin filament regulation. The results have been interpreted in terms of the crystallographic structure of troponin C and related to results with analogous calmodulin mutants.
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PMID:Modified calcium-dependent regulatory function of troponin C central helix mutants. 182 2

Limited proteolysis of gizzard myosin by alpha-chymotrypsin converted the heavy chain doublet pattern, seen by gel electrophoresis, to a single band. Light chain degradation was not observed and only minor cleavage occurred at other heavy chain sites. Using a polyclonal antibody raised against a unique sequence from the slower-migrating heavy chain (SM1) it was shown that this conversion was due to the loss of a peptide approximately 4000 daltons from the C terminus of SM1. The peptide was isolated and sequenced, and the cleavage site was identified between phenylalanine 1943 and alanine 1944. Addition of antibody before protease protected SM1 from cleavage. The following changes were observed (a) the Mg2(+)-dependence of actin-activated ATPase of digested phosphorylated myosin was altered and activity was relatively high at low Mg2+ levels, i.e. similar to phosphorylated heavy meromyosin; (b) the KCl dependence of Mg2(+)-ATPase of the digested myosin, particularly the phosphorylated form, showed an altered pattern consistent with the stabilization of the 6 S conformation; (c) the tendency for aggregation was increased by proteolysis of phosphorylated myosin. These results show that the C-terminal region of a gizzard myosin heavy chain can modify some of the properties of myosin. It is suggested that the observed modifications reflect an enhanced tendency of the digested myosin to aggregate.
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PMID:Cleavage of a smooth muscle myosin heavy chain near its C terminus by alpha-chymotrypsin. Effect on the properties of myosin. 182 82

UvrA is the ATPase subunit of the DNA repair enzyme (A)BC excinuclease. The amino acid sequence of this protein has revealed, in addition to two zinc fingers, three pairs of nucleotide binding motifs each consisting of a Walker A and B sequence. We have conducted site-specific mutagenesis, ATPase kinetic analyses, and nucleotide binding equilibrium measurements to correlate these sequence motifs with activity. Replacement of the invariant Lys by Ala in the putative A sequences indicated that K37 and K646 but not K353 are involved in ATP hydrolysis. In contrast, substitution of the invariant Asp by Asn in the B sequences at positions D238, D513, or D857 had little effect on the in vivo activity of the protein. Nucleotide binding studies revealed a stoichiometry of 0.5 ADP/UvrA monomer while kinetic measurements on wild-type and mutant proteins showed that the active form of UvrA is a dimer with 2 catalytic sites which interact in a positive cooperative manner in the presence of ADP; mutagenesis of K37 but not of K646 attenuated this cooperativity. Loss of ATPase activity was about 75% in the K37A, 86% in the K646A mutant, and 95% in the K37A-K646A double mutant. These amino acid substitutions had only a marginal effect on the specific binding of UvrA to damaged DNA but drastically reduced its ability to deliver UvrB to the damage site. We find that the deficient UvrB loading activity of these mutant UvrA proteins results from their inability to associate with UvrB in the form of (UvrA)2(UvrB)1 complexes. We conclude that UvrA forms a dimer with two ATPase domains involving K37 and K646 and that the work performed by ATP hydrolysis is the delivery of UvrB to the damage site on DNA.
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PMID:Site-specific mutagenesis of conserved residues within Walker A and B sequences of Escherichia coli UvrA protein. 182 50

Site-specific mutagenesis of the sarcoplasmic reticulum Ca(2+)-ATPase was used to investigate the functional roles of 18 amino acid residues located at or near the "hinge-domain," a highly conserved region of the cation-transporting ATPases. Mutation of Lys684 to arginine, alanine, histidine, and glutamine resulted in complete loss of calcium transport function and ATPase activity. For the Lys684----Ala, histidine, and glutamine mutants, this coincided with a loss of the ability to form a phosphorylated intermediate from ATP or Pi. The Lys684----Arg mutant retained the ability to phorphorylate from ATP with normal apparent affinity, demonstrating the importance of the positive charge. On the other hand, no phosphorylation was observed with Pi as substrate in this mutant. Examination of the partial reactions after phosphorylation from ATP in the Lys684----Arg mutant demonstrated a reduction of the rate of transformation of the ADP-sensitive phosphoenzyme intermediate (E1P) to the ADP-insensitive phosphoenzyme intermediate (E2P), which could account for the loss of transport function. Once accumulated, the E2P intermediate was able to decompose rapidly in the presence of K+ at neutral pH. These results may be interpreted in terms of a preferential destabilization of protein phosphate interactions in the E2P form of this mutant. The Asp703----Ala and Asn-Asp707----Ala-Ala mutants were completely inactive and unable to form phosphoenzyme intermediates from ATP or Pi. In these mutants as well as in the Lys684----Ala mutant, nucleotides were found to protect with normal affinity against intramolecular cross-linking induced with glutaraldehyde, indicating that the nucleotide binding site was intact. Mutation of Glu646, Glu647, Asp659, Asp660, Glu689, Asp695, Glu696, Glu715, and Glu732 to alanine did not affect the maximum rates of calcium transport and ATP hydrolysis or the apparent affinities for calcium and ATP. Mutation of the 2 highly conserved proline residues, Pro681 and Pro709, as well as Lys728, to alanine resulted in partially inhibited Ca(2+)-ATPase enzymes with retention of the ability to form a phosphoenzyme intermediate from ATP or Pi and with normal apparent affinities for ATP and calcium. The proline mutants retained the biphasic ATP concentration dependence of ATPase activity, characteristic of the wild-type, and therefore the partial inhibition of turnover could not be ascribed to a disruption of the low affinity modulatory ATP site.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Functional consequences of alterations to amino acids located in the hinge domain of the Ca(2+)-ATPase of sarcoplasmic reticulum. 183 54

The yeast plasma membrane H+-ATPase is activated in vivo by glucose metabolism, and previous deletion analysis has shown the C-terminus of the enzyme to be involved in this regulation. Site-directed mutagenesis demonstrates that Arg909 and Thr912 at the C-terminus are important for the increase in Vmax of the ATPase induced by glucose. Other changes in kinetic parameters induced by glucose are largely independent of these amino acids. Arg909 and Thr912 form a potential phosphorylation site for calmodulin-dependent multiprotein kinase. A double mutation of Ser911 and Thr912 to Ala results in no cell growth in glucose medium and greatly reduced activation of the ATPase by glucose. Growth and activity are restored by a third mutation (Ala547----Val) at the catalytic domain, providing genetic evidence for domain interaction.
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PMID:Analysis of the regulatory domain of yeast plasma membrane H+-ATPase by directed mutagenesis and intragenic suppression. 183 68

A sequence motif in the beta subunit of Escherichia coli F1 (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residue 149-156, where conserved residues are underlined) is one of the glycine-rich sequences found in many nucleotide binding proteins. In this study, we constructed a plasmid carrying all the F0F1 genes. This plasmid gave the highest membrane ATPase activity so far reported. Substitution of beta Gly149 by Ser suppressed the effect of the beta Ser174----Phe mutation (defective H(+)-ATPase), but beta Gly150----Ser substitution did not have this effect. A single mutation (beta Gly149----Ser or beta Gly150----Ser) gave active enzyme with altered divalent cation dependency and azide sensitivity: the beta Gly149----Ser mutant enzyme had 100-fold lower azide sensitivity and essentially no Ca(2+)-dependent activity, but had the wild-type level of Mg(2+)-dependent activity with active oxidative phosphorylation. Introduction of a beta Gly149----Ser or beta Gly150----Ser mutation with the beta Ser174----Phe mutation also lowered the Ca(2+)-dependent activity and azide sensitivity. Consistent with our previous findings (Takeyama, M., Ihara, K., Moriyama, Y., Noumi, T., Ida, K., Tomioka, N., Itai, A., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 21279-21284), a beta Thr156----Ala or Cys mutation impaired ATPase activity, suggesting that the hydroxyl moiety at position 156 is essential for the catalytic activity. The possible location of the catalytic site including divalent cation binding site(s) is discussed.
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PMID:Mutations in Ser174 and the glycine-rich sequence (Gly149, Gly150, and Thr156) in the beta subunit of Escherichia coli H(+)-ATPase. 183 55


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