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

---

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

The noncovalent fluorescent probe 6-propionyl-2-(dimethylamino)naphthalene (prodan) binds stoichiometrically to myosin subfragment-1 (S-1) without affecting the ATPase and actin-binding properties of S-1. Neither ATP nor actin interferes with the prodan binding. Free prodan exhibits a green emission peak at 520 nm. However, the prodan bound to S-1 and the S-1.ADP complex shows blue emission peaks at 460 and 450 nm, respectively, which allow easy separation of the fluorescence contributions from the free and bound probes. In the S-1.ADP.Pi state, the blue emission peak is further shifted to 445 nm with a large (4.5-fold) fluorescence enhancement. Thus, prodan in the presence of S-1 exhibits predominantly blue fluorescence only during ATP hydrolysis, and so visualizes the ATPase reaction continuously. The initial velocities of the steady state of the Mg2+-, Ca2+-, and actin-activated ATPases can be conveniently calculated from the blue fluorescence changes. The ability of different nucleoside triphosphates (NTP) to enhance the blue fluorescence of prodan follows the order ATP > CTP > UTP > ITP > GTP. This order agrees with those of the extent of hydrophobicity near the ribose of the corresponding nucleoside diphosphates (NDP) trapped to S-1 with orthovanadate (Vi) [Hiratsuka, T. (1984) J. Biochem. (Tokyo) 96, 155-162] and the ability of different NTPs to support force production in muscle fibers [Regnier, M., et al. (1993) Biophys. J. 64, A250]. The rate of formation of the corresponding S-1.NDP.Vi complex also follows this order, whereas the NTPase rate follows the reverse order. These results indicate that nucleotide-induced changes in prodan fluorescence correspond to the nucleotide-induced conformational states of S-1. Thus, the use of prodan in studies of the myosin ATPase offers a new and promising approach not only to monitoring the ATPase reaction but also to investigating the structural changes during ATP hydrolysis.
...
PMID:Prodan fluorescence reflects differences in nucleotide-induced conformational states in the myosin head and allows continuous visualization of the ATPase reactions. 958 28

Cdr1p, an ATP-binding cassette transporter from the pathogenic yeast Candida albicans, confers resistance to several unrelated drugs including anti-Candida drugs (Prasad et al., 1995b). We demonstrate that the deletion of 237 bp (79 aa) from the 3' end of CDR1 (which encompasses the transmembrane domain (TM) 12 of the putative transporter) did not result in the total loss of its ability to efflux cytotoxic agents. While the expression of deltaCDR1 in yeast resulted in impaired sensitivity to drugs like cycloheximide, anisomycin, sulfomethuron methyl and antifungal nystatin, its ability to confer resistance remained unaltered to drugs such as o-phenanthroline, 4-nitroquinoline-N-oxide, cerulenin, azoles, oligomycin, erythromycin, and benomyl. Similar to human MDR1p. Cdr1p might also have localized drug binding sites in TM 12, but that might not be the case for all the drugs. The TM 12 deletion also did not lead to any significant impairment in NTPase activities. Both ATPase and UTPase activities of complete Cdr1p and deltaCdr1p were not significantly altered, as was the case with respect to their ability to efflux Rh123 and steroid hormone like [3H]-beta-estradiol. To further dissect the functionality of Cdr1p, its truncated version was overexpressed in a baculovirus-insect cell expression system. The synthesis of deltaCdr1p in Sf9 cells was temporally regulated as a function of the baculovirus polyhedrin gene promoter. The Sf9 derived deltaCdr1p was approximately 130 kDa, which was lower than the expected size, probably due to the differences in glycosylation. This, however, did not affect the functionality of deltaCdr1p. The deletion of TM 12 did not affect the targeting of the protein and deltaCdr1p was exclusively localized in plasma membrane of Sf9 cells as detected by immunofluorescence. The expression of deltaCdr1p in the baculovirus-insect expression system generated a high drug-stimulated plasma membrane-bound ATPase activity which was not demonstrable when deltaCdr1p was expressed in yeast.
...
PMID:Deletion of transmembrane domain 12 of CDR1, a multidrug transporter from Candida albicans, leads to altered drug specificity: expression of a yeast multidrug transporter in baculovirus expression system. 960 4

The mechanical behavior of skinned rabbit psoas muscle fiber contractions and in vitro motility of F-actin (Vf) have been examined using ATP, CTP, UTP, or their 2-deoxy forms (collectively designated as nucleotide triphosphates or NTPs) as contractile substrates. Measurements of actin-activated heavy meromyosin (HMM) NTPase, the rates of NTP binding to myosin and actomyosin, NTP-mediated acto-HMM dissociation, and NTP hydrolysis by acto-HMM were made for comparison to the mechanical results. The data suggest a very similar mechanism of acto-HMM NTP hydrolysis. Whereas all NTPs studied support force production and stiffness that vary by a factor 2 or less, the unloaded shortening velocity (Vu) of muscle fibers varies by almost 10-fold. 2-Deoxy ATP (dATP) was unique in that Vu was 30% greater than with ATP. Parallel behavior was observed between Vf and the steady-state maximum actin-activated HMM ATPase rate. Further comparisons suggest that the variation in force correlates with the rate and equilibrium constant for NTP cleavage; the variations in Vu or Vf are related to the rate of cross-bridge dissociation caused by NTP binding or to the rate(s) of product release.
...
PMID:ATP analogs and muscle contraction: mechanics and kinetics of nucleoside triphosphate binding and hydrolysis. 963 59

In the maturation of the Escherichia coli antibiotic Microcin B17, the product of the mcbA gene is modified posttranslationally by the multimeric Microcin synthetase complex (composed of McbB, C, and D) to cyclize four Cys and four Ser residues to four thiazoles and four oxazoles, respectively. The purified synthetase shows an absolute requirement for ATP or GTP in peptide substrate heterocyclization, with GTP one-third as effective as ATP in initial rate studies. The ATPase/GTPase activity of the synthetase complex is conditional in that ADP or GDP formation requires the presence of substrate; noncyclizable versions of McbA bind to synthetase, but do not induce the NTPase activity. The stoichiometry of ATP hydrolysis and heterocycle formation is 5:1 for a substrate that contains two potential sites of modification. However, at high substrate concentrations (>50Km) heterocycle formation is inhibited, while ATPase activity occurs undiminished, consistent with uncoupling of NTP hydrolysis and heterocycle formation at high substrate concentrations. Sequence homology reveals that the McbD subunit has motifs reminiscent of the Walker B box in ATP utilizing enzymes and of motifs found in small G protein GTPases. Mutagenesis of three aspartates to alanine in these motifs (D132, D147, and D199) reduced Microcin B17 production in vivo and heterocycle formation in vitro, suggesting that the 45 kDa McbD has a regulated ATPase/GTPase domain in its N-terminal region necessary for peptide heterocyclization.
...
PMID:ATP/GTP hydrolysis is required for oxazole and thiazole biosynthesis in the peptide antibiotic microcin B17. 974 32

Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH-I) is a DNA-dependent ATPase that serves as a transcription termination factor during viral mRNA synthesis. NPH-I is a member of the DExH box family of nucleic acid-dependent nucleoside triphosphatases (NTPases), which is defined by the presence of several conserved sequence motifs. We have assessed the contributions of individual amino acids (underlined) in motifs I (GxGKT), II (DExHN), III (SAT), and VI (QxxGRxxR) to ATP hydrolysis by performing alanine scanning mutagenesis. Significant decrements in ATPase activity resulted from mutations at nine positions: Lys-61 and Thr-62 (motif I); Asp-141, Glu-142, His-144, and Asn-145 (motif II); and Gln-472, Arg-476, and Arg-479 (motif VI). Structure-function relationships at each of these positions were clarified by introducing conservative substitutions and by steady-state kinetic analysis of the mutant enzymes. Comparison of our findings for NPH-I with those of mutational studies of other DExH and DEAD box proteins underscores similarities as well as numerous disparities in structure-activity relationships. We conclude that the functions of the conserved amino acids of the NTPase motifs are context dependent.
...
PMID:Mutational analysis of vaccinia virus nucleoside triphosphate phosphohydrolase I, a DNA-dependent ATPase of the DExH box family. 988 35

Hepatitis G virus (HGV) nonstructural protein 3 (NS3) contains amino acid sequence motifs typical of ATPase and RNA helicase proteins. In order to examine the RNA helicase activity of the HGV NS3 protein, the NS3 region (amino acids 904 to 1580) was fused with maltose-binding protein (MBP), and the fusion protein was expressed in Escherichia coli and purified with amylose resin and anion-exchange chromatography. The purified MBP-HGV/NS3 protein possessed RNA-stimulated ATPase and RNA helicase activities. Characterization of the ATPase and RNA helicase activities of MBP-HGV/NS3 showed that the optimal reaction conditions were similar to those of other Flaviviridae viral NS3 proteins. However, the kinetic analysis of NTPase activity showed that the MBP-HGV/NS3 protein had several unique properties compared to the other Flaviviridae NS3 proteins. The HGV NS3 helicase unwinds RNA-RNA duplexes in a 3'-to-5' direction and can unwind RNA-DNA heteroduplexes and DNA-DNA duplexes as well. In a gel retardation assay, the MBP-HGV/NS3 helicase bound to RNA, RNA/DNA, and DNA duplexes with 5' and 3' overhangs but not to blunt-ended RNA duplexes. We also found that the conserved motif VI was important for RNA binding. Further deletion mapping showed that the RNA binding domain was located between residues 1383 and 1395, QRRGRTGRGRSGR. Our data showed that the MBP-HCV/NS3 protein also contains the RNA binding domain in the similar domain.
...
PMID:RNA-Stimulated ATPase and RNA helicase activities and RNA binding domain of hepatitis G virus nonstructural protein 3. 1007 39

The hepatitis C virus nonstructural 3 protein (NS3) possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion. The serine protease activity is required for proteolytic processing at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B polyprotein cleavage sites. NS3 forms a complex with NS4A, a 54-residue polypeptide that was shown to act as an essential cofactor of the NS3 protease. We have expressed in Escherichia coli the NS3-NS4A precursor; cleavage at the junction between NS3 and NS4A occurs during expression in the bacteria cells, resulting in the formation of a soluble noncovalent complex with a sub-nanomolar dissociation constant. We have assessed the minimal ionic strength and detergent and glycerol concentrations required for maximal proteolytic activity and stability of the purified NS3-NS4A complex. Using a peptide substrate derived from the NS5A-NS5B junction, the catalytic efficiency (kcat/Km) of NS3-NS4A-associated protease under optimized conditions was 55 000 s-1 M-1, very similar to that measured with a recombinant complex purified from eukaryotic cells. Dissociation of the NS3-NS4A complex was found to be fully reversible. No helicase activity was exhibited by the purified NS3-NS4A complex, but NS3 was fully active as a helicase upon dissociation of NS4A. On the other hand, both basal and poly(U)-induced NTPase activity and ssRNA binding activity associated with the NS3-NS4A complex were very similar to those exhibited by NS3 alone. Therefore, NS4A appears to uncouple the ATPase/ssRNA binding and RNA unwinding activities associated with NS3.
...
PMID:Modulation of hepatitis C virus NS3 protease and helicase activities through the interaction with NS4A. 1022 Mar 51

A human brain E-type ATPase (HB6 ecto-apyrase) was subjected to site-directed mutagenesis to assess the functional significance of two highly conserved tryptophan residues (Trp 187 and Trp 459), the only two tryptophans conserved in nearly all E-type ATPases. Mutation of tryptophan 187 to alanine yielded a poorly expressed ecto-apyrase completely devoid of nucleotidase activity. Immunolocalization of the W187A mutant in mammalian COS cells showed a cellular distribution clearly different from that of the wild-type enzyme, with the majority of the immunoreactivity concentrated in the interior of the cell. Unlike the wild-type enzyme, this mutant did not bind the nucleotide analogue Cibacron Blue and was sensitive to proteolytic digestion by chymotrypsin. These results suggest alteration of the tertiary structure, causing the enzyme to be improperly folded and retained within the cell. In contrast, mutation of tryptophan 459 to alanine resulted in an ecto-apyrase with enhanced NTPase activity, but diminished NDPase activity. Immunolocalization of this active mutant ecto-apyrase revealed a cellular pattern similar to that of the wild-type enzyme, distributed along the cell periphery and in cell processes. Coupling this active W459A mutation to a previously described mutation (D219E) resulted in an enzyme which preferentially hydrolyzes nucleoside triphosphates over diphosphates. The D219E/W459A double mutant had an ATPase:ADPase ratio of 11:1 and a UTPase:UDPase ratio of 148:1. In addition, the double mutant is substantially less sensitive to inhibition by azide, a more potent inhibitor of ecto-apyrases than ecto-ATPases. Thus, mutation of only two amino acids of an E-type ATPase essentially converts an ecto-apyrase to an ecto-NTPase.
...
PMID:Mutagenesis of two conserved tryptophan residues of the E-type ATPases: inactivation and conversion of an ecto-apyrase to an ecto-NTPase. 1023 36

Saccharomyces cerevisiae Cet1p is the prototype of a family of metal-dependent RNA 5'-triphosphatases/NTPases encoded by fungi and DNA viruses; the family is defined by conserved sequence motifs A, B, and C. We tested the effects of 12 alanine substitutions and 16 conservative modifications at 18 positions of the motifs. Eight residues were identified as important for triphosphatase activity. These were Glu-305, Glu-307, and Phe-310 in motif A (IELEMKF); Arg-454 and Lys-456 in motif B (RTK); Glu-492, Glu-494, and Glu-496 in motif C (EVELE). Four acidic residues, Glu-305, Glu-307, Glu-494, and Glu-496, may comprise the metal-binding site(s), insofar as their replacement by glutamine inactivated Cet1p. E492Q retained triphosphatase activity. Basic residues Arg-454 and Lys-456 in motif B are implicated in binding to the 5'-triphosphate. Changing Arg-454 to alanine or glutamine resulted in a 30-fold increase in the K(m) for ATP, whereas substitution with lysine increased K(m) 6-fold. Changing Lys-456 to alanine or glutamine increased K(m) an order of magnitude; ATP binding was restored when arginine was introduced. Alanine in lieu of Phe-310 inactivated Cet1p, whereas Tyr or Leu restored function. Alanine mutations at aliphatic residues Leu-306, Val-493, and Leu-495 resulted in thermal instability in vivo and in vitro. A second S. cerevisiae RNA triphosphatase/NTPase (named Cth1p) containing motifs A, B, and C was identified and characterized. Cth1p activity was abolished by E87A and E89A mutations in motif A. Cth1p is nonessential for yeast growth and, by itself, cannot fulfill the essential role played by Cet1p in vivo. Yet, fusion of Cth1p in cis to the guanylyltransferase domain of mammalian capping enzyme allowed Cth1p to complement growth of cet1Delta yeast cells. This finding illustrates that mammalian guanylyltransferase can be used as a vehicle to deliver enzymes to nascent pre-mRNAs in vivo, most likely through its binding to the phosphorylated CTD of RNA polymerase II.
...
PMID:Mutational analyses of yeast RNA triphosphatases highlight a common mechanism of metal-dependent NTP hydrolysis and a means of targeting enzymes to pre-mRNAs in vivo by fusion to the guanylyltransferase component of the capping apparatus. 1050 29

Non-structural protein 2C is known to play a fundamental role in the replication of picornaviruses. Sequence analyses revealed that 2C belongs to a rapidly expanding group of proteins containing a consensus sequence for nucleotide binding (NTB). We report that echovirus 9 polypeptide 2C displays NTPase activity in vitro. In our experiments, several P2 genes were expressed in Escherichia coli as fusion proteins linked to glutathione S-transferase (GST) prior to purification close to homogeneity. In contrast to GST-2B, both GST-2C and GST-2BC showed ATPase as well as GTPase activity indicating that the site for NTB binding and splitting is located in 2C.
...
PMID:Echovirus 9 strain barty non-structural protein 2C has NTPase activity. 1058 88


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---