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:3.6.1.25 (triphosphatase)
1,529 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chlorella virus RNA triphosphatase (cvRtp1) is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, poxviruses, and baculoviruses. The primary structure of cvRtp1 is more similar to that of the yeast RNA triphosphatase Cet1 than it is to the RNA triphosphatases of other DNA viruses. To evaluate the higher order structural similarities between cvRtp1 and the fungal enzymes, we performed an alanine scan of individual residues of cvRtp1 that were predicted, on the basis of the crystal structure of Cet1, to be located at or near the active site. Twelve residues (Glu(24), Glu(26), Asp(64), Arg(76), Lys(90), Glu(112), Arg(127), Lys(129), Arg(131), Asp(142), Glu(163), and Glu(165)) were deemed essential for catalysis by cvRtp1, insofar as their replacement by alanine reduced phosphohydrolase activity to <5% of the wild-type value. Structure-activity relationships were elucidated by introducing conservative substitutions at the essential positions. The mutational results suggest that the active site of cvRtp1 is likely to adopt a tunnel fold like that of Cet1 and that a similar constellation of side chains within the tunnel is responsible for metal binding and reaction chemistry. Nonetheless, there are several discordant mutational effects in cvRtp1 versus Cet1, which suggest that different members of the phosphohydrolase family vary in their reliance on certain residues within the active site tunnel. We found that tripolyphosphate and pyrophosphate were potent competitive inhibitors of cvRtp1 (K(i) = 0.6 microm tripolyphosphate and 2.4 microm pyrophosphate, respectively), whereas phosphate had little effect. cvRtp1 displayed a weak intrinsic tripolyphosphatase activity (3% of its ATPase activity) but was unable to hydrolyze pyrophosphate.
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PMID:Chlorella virus RNA triphosphatase. Mutational analysis and mechanism of inhibition by tripolyphosphate. 1184 1

Compounds that interact with DNA or RNA generally act as inhibitors of enzymes that unwind DNA or RNA. In the present study we describe the synthesis and properties of some nucleoside analogues that interact with double-stranded DNA but that, in contrast, facilitate the unwinding reaction mediated by West Nile (WN) virus nucleoside triphosphatase (NTPase)/helicase. The nucleoside analogues described, 1-(2'-O-methyl-beta-D-ribofuranosyl)imidazo[4,5-d]pyridazine-4,7(5H,6H)-dione (HMC-HO4), 1-(beta-D-ribofuranosyl)imidazo[4,5-d]pyridazine-4,7(5H,6H)-dione, and 1-(2'-deoxy-alpha-D-ribofuranosyl)imidazo[4,5-d]pyridazine-4,7(5H,6H)dione, all contain the imidazo[4,5-d]pyridazine ring system. The extent of the enhancing effect on helicase activity was found to be dependent on the time of exposure of the DNA substrate to the compounds and their concentrations. The nucleoside analogues were nevertheless found to be capable of uncoupling the ATPase and helicase activities of the enzyme by a mechanism operating on the level of the enzyme. Thus, in the case of HMC-HO4, the direct interaction with the enzyme caused inhibition of its helicase activity, with a half-maximal inhibitory concentration of 30 microM. The similar potency of the compound against replication of WN virus in cell culture suggests that inhibition of the helicase activity of the viral enzyme is responsible for the observed antiviral activity of HMC-HO4 and may indeed represent an important mode of action of antiviral drugs in general. Comparative studies performed with the related NTPase/helicase from hepatitis C virus revealed that the extent of the effects mediated by imidazo[4,5-d]pyridazine nucleosides is enzyme specific. The substances described may represent a starting point for the development of a new class of helicase-specific antivirals.
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PMID:Characterization of imidazo[4,5-d]pyridazine nucleosides as modulators of unwinding reaction mediated by West Nile virus nucleoside triphosphatase/helicase: evidence for activity on the level of substrate and/or enzyme. 1195 50

The multidrug resistance proteins P-glycoprotein (Pgp) and MRP1 are drug-efflux pumps. In this study, we compared the nucleotide triphosphatase activities of the isolated N-terminal nucleotide binding domains (NBD1) of Pgp and MRP1, and explored the potential role of the phosphorylation target domain of Pgp on the regulation of Pgp NBD1 ATPase activity. We found that: (1) the NBD1s of Pgp and MRP1 have ATPase and GTPase activities, (2) the K(m)s of Pgp NBD1 for ATP and GTP hydrolysis are identical, while the K(m) of MRP1 NBD1 for ATP is lower than that for GTP, and (3) phosphorylation of MLD by PKA or PKC produces a marginal increase of V(max) for ATP hydrolysis, without affecting the affinity for ATP. These results show efficient GTP hydrolysis by the NBD1s of Pgp and MRP1, and a minor role of phosphorylation in the control of Pgp NBD1 ATPase activity.
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PMID:Nucleotide triphosphatase activity of the N-terminal nucleotide-binding domains of the multidrug resistance proteins P-glycoprotein and MRP1. 1216 30

Late gene expression factor 4 (LEF4), a multifunctional protein encoded by the Bombyx mori nucleopolyhedrovirus has been bacterially expressed and characterized. Sequence analyses and three-dimensional modelling of B. mori LEF4 showed that the protein is related to mRNA-capping enzymes, which are organized as two modular domains. Most of the acidic side chains in LEF4 were solvent-exposed and spread all along the fold. A region dominated by negatively charged groups, which protrudes from the larger domain was ideally suited for interactions with proteins having positively charged patches at the surface. The purified LEF4 protein exhibited different enzyme activities associated with mRNA-capping enzymes, i.e. GTP-binding, RNA triphosphatase and guanylate transferase activities. In addition, LEF4 also showed NTP-hydrolysing activity. The kinetic analysis of ATP hydrolysis revealed a sigmoidal response with two deduced binding sites for ATP, whereas the guanylate transferase activity showed a typical hyperbolic response to varying concentrations of GTP with a Km of 330+/-20 microM. Analysis of the modelled three-dimensional structure of LEF4 suggested the presence of crucial residues in sequence motifs important for the integrity of the fold. Mutation of one such conserved and buried tyrosine residue to cysteine in the motif IIIa, located close to the interlobe region of the model, resulted in a 44% loss of guanylate transferase activity of LEF4 but had no effect on the ATPase activity.
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PMID:Functional characterization and structural modelling of late gene expression factor 4 from Bombyx mori nucleopolyhedrovirus. 1216 97

The present study describes the localization and distribution of putative ecto-nucleoside-triphosphate-diphosphohydrolases in the frog semicircular canals. These enzymes provide the terminating mechanism of adenosine-5'-triphosphate (ATP) signalling. The localization of the ATP hydrolysis was mapped ultracytochemically using a one-step cerium citrate reaction. Electron-dense precipitates, indicating ecto-adenosine-triphosphatase (ecto-ATPase) activity, were found at the outer surface of plasma membranes of crista hair cells and supporting cells of the sensory epithelium, transitional cells and undifferentiated cells of the ampullar wall and dark cells constituting the secretory epithelium. Non-sensory cells of the ampulla usually exhibited reaction deposits at the level of both apical and basolateral membranes coming into contact with the endolymph and the perilymph respectively, while cells constituting the sensory epithelium showed evident differences in relation to their position. Hair cells and supporting cells of the peripheral regions exhibited clear reaction products both at the level of apical and basolateral membranes, while those of the isthmus region showed abundant reactivity only at the level of their apical membranes. Of particular interest was the observation that hair cell stereocilia exhibited an abundant ecto-ATPase activity, thus suggesting a possible colocalization of enzymatic sites with purinergic receptors and mechanotransduction channels. This strategic expression of ecto-ATPase sites could provide a rapid mechanism of ATP removal able to rapidly restore the sensitivity of transduction channels. In conclusion, the widespread distribution of ecto-ATPase sites at the level of sensory and non-sensory cells of the frog semicircular canals suggests that ATP may have a key role in controlling vestibular function.
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PMID:Ecto-ATPase activity sites in vestibular tissues: an ultracytochemical study in frog semicircular canals. 1258 76

The Candida drug resistance protein Cdr1p (approximately 170 kDa) is a member of ATP binding cassette (ABC) superfamily of drug transporters, characterized by the presence of 2 nucleotide binding domains (NBD) and 12 transmembrane segments (TMS). NBDs of these transporters are the hub of ATP hydrolysis activity, and their sequence contains a conserved Walker A motif (GxxGxGKS/T). Mutations of the lysine residue within this motif abrogate the ability of NBDs to hydrolyze ATP. Interestingly, the sequence alignments of Cdr1p NBDs with other bacterial and eukaryotic transporters reveal that its N-terminal NBD contains an unusual Walker A sequence (GRPGAGCST), as the invariant lysine is replaced by a cysteine. In an attempt to understand the significance of this uncommon positioning of cysteine within the Walker A motif, we for the first time have purified and characterized the N-terminal NBD (encompassing first N-terminal 512 amino acids) of Cdr1p as well as its C193A mutant protein. The purified NBD-512 protein could exist as an independent functional general ribonucleoside triphosphatase with strong divalent cation dependence. It exhibited ATPase activity with an apparent K(m) in the 0.8-1.0 mM range and V(max) in the range of 147-160 nmol min(-)(1) (mg of protein)(-)(1). NBD-512-associated ATPase activity was also sensitive to inhibitors such as vanadate, azide, and NEM. The Mut-NBD-512 protein (C193A) showed a severe impairment in its ability to hydrolyze ATP (95%); however, no significant effect on ATP (TNP-ATP) binding was observed. Our results show that C193 is critical for N-terminal NBD-mediated ATP hydrolysis and represents a unique feature distinguishing the ATP-dependent functionality of the ABC transporters of fungi from those found in bacteria and other eukaryotes.
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PMID:Purification and characterization of the N-terminal nucleotide binding domain of an ABC drug transporter of Candida albicans: uncommon cysteine 193 of Walker A is critical for ATP hydrolysis. 1296 7

Kidneys from winter bats (Myotis lucifugus) were removed and fixed in cold formalin-calcium while the animals were in the following states: (a) natural hibernation; (b) arousal from hibernation for 24 hours; (c) laboratory maintained hibernation; and (d) no hibernation since the previous winter. With fixed frozen sections, the lead salt method of Wachstein and Meisel with adenosine triphosphate as substrate (pH 7.2) showed enzymic activity localized in large vacuoles and smaller vesicles or droplets in the Golgi region of distal and proximal tubular epithelial cells of kidneys from hibernating bats. No ATPase activity was detected in the basal lamellae of tubular epithelium from hibernating bats. ATPase activity in the Golgi region was not seen in cells from kidney tubules of bats aroused from hibernation 24 hours previously or of animals that had not hibernated, whereas activity for ATPase was present in the basal infoldings of tubular epithelium from these animals. Inosine di- and triphosphatase and calcium activated ATPase activities were also detected in the Golgi region of hibernating bats but were not present in the basal infoldings of tubular epithelium from active animals. There was little or no activity toward the mono- and diphosphates of adenine, thiamine pyrophosphate, and the di- or triphosphates of guanidine, cytidine, or deoxyadenosine. The loss of enzymic activity from the Golgi region of the tubular epithelium from hibernating bats and its increase in the region of the basal infoldings of tubular epithelium in aroused bats suggests that the Golgi region plays a role in the synthesis of enzymic protein usually identified with the external cell membrane.
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PMID:CYTOCHEMICAL DIFFERENCES IN KIDNEYS FROM WINTER HIBERNATING AND AROUSED BATS (MYOTIS LUCIFUGUS), WITH PARTICULAR REFERENCE TO THE GOLGI ZONE. 1418 9

Many complex viruses acquire their genome by active packaging into a viral precursor particle called a procapsid. Packaging is performed by a viral portal complex, which couples ATP hydrolysis to translocation of nucleic acid into the procapsid. The packaging process has been studied for a variety of viruses, but the mechanism of the associated ATPase remains elusive. In this study, the mechanism of RNA translocation in double-stranded RNA bacteriophages is characterized using rapid kinetic analyses. The portal complex of bacteriophage 8 is a hexamer of protein P4, which exhibits nucleotide triphosphatase activity. The kinetics of ATP binding reveals a two-step process: an initial, fast, second-order association, followed by a slower, first-order phase. The slower phase exhibits a high activation energy and has been assigned to a conformational change. ATP binding becomes cooperative in the presence of RNA. Steady-state kinetics of ATP hydrolysis, which proceeds only in the presence of RNA, also exhibits cooperativity. On the other hand, ADP release is fast and RNA-independent. The steady-state rate of hydrolysis increases with the length of the RNA substrate indicating processive translocation. Raman spectroscopy reveals that RNA binds to P4 via the phosphate backbone. The ATP-induced conformational change affects the backbone of the bound RNA but leaves the protein secondary structure unchanged. This is consistent with a model in which cooperativity is induced by an RNA link between subunits of the hexamers and translocation is effected by an axial movement of the subunits relative to one another upon ATP binding.
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PMID:Enzymatic mechanism of RNA translocation in double-stranded RNA bacteriophages. 1453 Feb 66

During cap enameloid formation in gars (Lepisosteus oculatus), the dental epithelial cells that constitute the enamel organ were observed by means of transmission electron microscopy and enzyme cytochemistry to detect the hydrolytic enzyme activities, alkaline phosphatase (ALPase), acid phosphatase (ACPase), calcium-dependent adenosine triphosphatase (Ca-ATPase) and potassium-dependent p-nitrophenylphosphatase (K-NPPase) (sodium, potassium-activated adenoshine triphosphatase (Na-K-ATPase)). The enameloid formation process in gars was divided into three stages: matrix formation, mineralisation and maturation. The enamel organ consisted of the outer dental epithelial (ODE) cells, stellate reticulum (SR), stratum intermedium (SI) and the inner dental epithelial (IDE) cells during the whole of the cap enameloid formation stages. During the matrix formation stage, many cisternae of rough endoplasmic reticulum and widely distributed Golgi apparatus, in which the procollagen granules containing cross-striations were often found, were remarkable elements in the IDE cells. During the stage of mineralisation, the IDE cells were tall columnar, and infoldings of distal plasma membrane of the IDE cells became marked. The most developed Golgi apparatus was visible at this stage, and large secretory granules containing fine granular or tubular materials were found in the distal cytoplasm that was close to the infoldings of the distal end. Many lysosomes that were ACPase positive were seen near the Golgi apparatus and in the distal cytoplasm of the IDE cells. ACPase positive granules often contained the cross-striation structure resembling procollagen, suggesting that the procollagen is degenerated in the IDE cells. During the maturation stage, the distal infoldings became unclear, and there were no large granules containing tubular materials, but many ACPase positive lysosomes were still present in the IDE cells. Non-specific ALPase was detected at the plasma membrane of the IDE cells at the mineralisation and maturation stages. K-NPPase was markedly detected at the plasma membrane of the IDE cells at the maturation stage. These results demonstrate that the IDE cells might be mainly involved in the removal of degenerated organic matrix from enameloid during the later formation stages. Strong Ca-ATPase activity was observed at the entire plasma membrane of the stratum intermedium cells, and there was slightly weak activity at the plasma membrane of the IDE cells during the mineralisation and maturation stages, implying that these cells are related to the active Ca transport to the maturing enameloid. It is likely that although the structure of the enamel organ is different, the function, especially at the mineralisation and maturation stages, is similar to other actinopterygians having well-mineralized cap enameloid.
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PMID:Fine structural and cytochemical mapping of enamel organ during the enameloid formation stages in gars, Lepisosteus oculatus, Actinopterygii. 1574 91

The incision of damaged DNA by the Escherichia coli UvrABC endonuclease requires ATP hydrolysis. Although the deduced sequence of the UvrB protein suggests a putative ATP binding site, no nucleoside triphosphatase activity is demonstrable with the purified UvrB protein. The UvrB protein is specifically proteolyzed in E. coli cell extracts to yield a 70 kD fragment, referred to as UvrB*, which has been purified and is shown to possess a single-strand DNA dependent ATPase activity. Substrate specificity and kinetic analyses of UvrB* catalyzed nucleotide hydrolysis indicate that the stimulation in DNA dependent ATPase activity following formation of the UvrAB complex results from the activation of the normally sequestered UvrB associated ATPase. Using nucleotide analogues, it can be shown that this activity is essential to the DNA incision reaction carried out by the UvrABC complex.
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PMID:Involvement of a cryptic ATPase activity of UvrB and its proteolysis product, UvrB* in DNA repair. 297 38


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