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)

COS-7 cells were transiently transfected with human thyrotropin receptor and dog A1 adenosine receptor cDNAs. An A1 agonist, N6-(L-2-phenylisopropyl) adenosine (PIA), which is ineffective alone, enhanced the thyrotropin (TSH)-induced inositol phosphate production, reflecting phospholipase C (PLC) activation, but inhibited the TSH-induced cAMP accumulation, reflecting adenylyl cyclase inhibition. These PIA-induced actions were completely inhibited by pertussis toxin (PTX) treatment. Moreover, in the cells expressing a PTX-insensitive mutant of Gi2alpha or Gi3alpha, in which a glycine residue was substituted for a cysteine residue to be ADP-ribosylated by PTX, at the fourth position of the C terminus, PIA effectively exerted both stimulatory and inhibitory effects on the TSH-induced actions although the cells were treated with the toxin. Overexpression of the betagamma subunits of the G proteins enhanced the TSH-induced inositol phosphate production without any significant effect on the cAMP response; under these conditions, PIA did not further increase the elevated inositol phosphate response to TSH. On the contrary, overexpression of a constitutively active mutant of Gi2alpha, in which the guanosine triphosphatase activity is lost, inhibited the TSH-induced cAMP accumulation but hardly affected the inositol phosphate response; under these conditions, PIA never exerted further inhibitory effects on the cAMP response to TSH. In contrast to the case of the TSH-induced inositol phosphate response, the response to a constitutively active G11alpha mutant was not appreciably affected, and that to NaF was rather inhibited by PIA and overexpression of the betagamma subunits. Taken together, these results suggest that a single type of PTX-sensitive G protein mediates the A1 adenosine receptor-linked modulation of two signaling pathways in collaboration with an activated thyrotropin receptor; alpha subunits of the PTX-sensitive G proteins mediate the inhibitory action on adenylyl cyclase, and the betagamma subunits mediate the stimulatory action on PLC. In the case of the latter stimulatory action on PLC, the betagamma subunits may not directly activate PLC. The possible mechanism by which betagamma subunits enhance the TSH-induced PLC activation is discussed.
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PMID:Betagamma subunits of pertussis toxin-sensitive G proteins mediate A1 adenosine receptor agonist-induced activation of phospholipase C in collaboration with thyrotropin. A novel stimulatory mechanism through the cross-talk of two types of receptors. 928 15

The 2.4-A resolution crystal structure of a dominantly active form of the small guanosine triphosphatase (GTPase) RhoA, RhoAV14, complexed with the nonhydrolyzable GTP analogue, guanosine 5'-3-O-(thio)triphosphate (GTPgammaS), reveals a fold similar to RhoA-GDP, which has been recently reported (Wei, Y., Zhang, Y., Derewenda, U., Liu, X., Minor, W., Nakamoto, R. K., Somlyo, A. V., Somlyo, A. P., and Derewenda, Z. S. (1997) Nat. Struct. Biol. 4, 699-703), but shows large conformational differences localized in switch I and switch II. These changes produce hydrophobic patches on the molecular surface of switch I, which has been suggested to be involved in its effector binding. Compared with H-Ras and other GTPases bound to GTP or GTP analogues, the significant conformational differences are located in regions involving switches I and II and part of the antiparallel beta-sheet between switches I and II. Key residues that produce these conformational differences were identified. In addition to these differences, RhoA contains four insertion or deletion sites with an extra helical subdomain that seems to be characteristic of members of the Rho family, including Rac1, but with several variations in details. These sites also display large displacements from those of H-Ras. The ADP-ribosylation residue, Asn41, by C3-like exoenzymes stacks on the indole ring of Trp58 with a hydrogen bond to the main chain of Glu40. The recognition of the guanosine moiety of GTPgammaS by the GTPase contains water-mediated hydrogen bonds, which seem to be common in the Rho family. These structural differences provide an insight into specific interaction sites with the effectors, as well as with modulators such as guanine nucleotide exchange factor (GEF) and guanine nucleotide dissociation inhibitor (GDI).
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PMID:Crystal structure of human RhoA in a dominantly active form complexed with a GTP analogue. 954 99

The human lymphoid cell activation antigen CD39 is a known E-type apyrase that hydrolyzes extracellular ATP and ADP, a function important in homotypic adhesion, platelet aggregation, and removal by activated lymphocytes of the lytic effect of ATP. The recently identified putative rat homologue of CD39L1 has been shown to have E-type ecto-ATPase activity, by hydrolyzing extracellular ATP. We have characterized three novel CD39-like transcripts, CD39L2, CD39L3, and CD39L4, which share extensive amino acid homology with other nucleotide triphosphatases in vertebrates, invertebrates, and plants, suggesting that these genes also encode proteins with ecto-nucleotidase activity. Isolation and sequencing of full-length cDNA clones for each gene identified putative proteins of 485, 529, and 429 amino acids. The expression pattern of all five human members of the gene family was analyzed. CD39L2, CD39L3, and CD39L4 were mapped on the human genome, and the murine homologues identified with the putative map locations were assigned on the basis of regions of conserved gene order between human and mouse chromosomes. The map location of mcd39l4 places the gene within a region associated with audiogenic seizure susceptibility in mouse. This disorder is characterized by convulsions induced by loud high-frequency sound and has been shown to be associated with increased nucleotide triphosphatase activity.
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PMID:The CD39-like gene family: identification of three new human members (CD39L2, CD39L3, and CD39L4), their murine homologues, and a member of the gene family from Drosophila melanogaster. 967 30

Autographa californica nuclear polyhedrosis virus (AcNPV) encodes a 168-amino-acid polypeptide that contains the signature motif of the superfamily of protein phosphatases that act via a covalent cysteinyl phosphate intermediate. The sequence of the AcNPV phosphatase is similar to that of the RNA triphosphatase domain of the metazoan cellular mRNA capping enzyme. Here, we show that the purified recombinant AcNPV protein is an RNA 5'-triphosphatase that hydrolyzes the gamma-phosphate of triphosphate-terminated poly(A); it also hydrolyzes ATP to ADP and GTP to GDP. The phosphatase sediments as two discrete components in a glycerol gradient: a 9.5S oligomer and 2.5S putative monomer. The 2.5S form of the enzyme releases 32Pi from 1 microM gamma-32P-labeled triphosphate-terminated poly(A) with a turnover number of 52 min-1 and converts ATP to ADP with Vmax of 8 min-1 and Km of 25 microM ATP. The 9.5S oligomeric form of the enzyme displays an initial pre-steady-state burst of ADP and Pi formation, which is proportional to and stoichiometric with the enzyme, followed by a slower steady-state rate of product formation (approximately 1/10 of the steady-state rate of the 2.5S enzyme). We surmise that the oligomeric enzyme is subject to a rate-limiting step other than reaction chemistry and that this step is either distinct from or slower than the rate-limiting step for the 2.5S enzyme. Replacing the presumptive active site nucleophile Cys-119 by alanine abrogates RNA triphosphatase and ATPase activity. Our findings raise the possibility that baculoviruses encode enzymes that cap the 5' ends of viral transcripts synthesized at late times postinfection by a virus-encoded RNA polymerase.
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PMID:Characterization of a baculovirus-encoded RNA 5'-triphosphatase. 969 98

Autographa californica nuclear polyhedrosis virus late and very late mRNAs are transcribed by an RNA polymerase consisting of four virus-encoded polypeptides: LEF-8, LEF-9, LEF-4, and p47. The 464-amino-acid LEF-4 subunit contains the signature motifs of GTP:RNA guanylyltransferases (capping enzymes). Here, we show that the purified recombinant LEF-4 protein catalyzes two reactions involved in RNA cap formation. LEF-4 is an RNA 5'-triphosphatase that hydrolyzes the gamma phosphate of triphosphate-terminated RNA and a guanylyltransferase that reacts with GTP to form a covalent protein-guanylate adduct. The RNA triphosphatase activity depends absolutely on a divalent cation; the cofactor requirement is satisfied by either magnesium or manganese. LEF-4 also hydrolyzes ATP to ADP and Pi (Km = 43 microM ATP; Vmax = 30 s-1) and GTP to GDP and Pi. The LEF-4 nucleoside triphosphatase (NTPase) is activated by manganese or cobalt but not by magnesium. The RNA triphosphatase and NTPase activities of baculovirus LEF-4 resemble those of the vaccinia virus and Saccharomyces cerevisiae mRNA capping enzymes. We suggest that these proteins comprise a novel family of metal-dependent triphosphatases.
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PMID:RNA 5'-triphosphatase, nucleoside triphosphatase, and guanylyltransferase activities of baculovirus LEF-4 protein. 981 40

Adducin is a membrane skeletal protein that binds to actin filaments (F-actin) and thereby promotes the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such as ruffling membranes. Rho-associated kinase (Rho- kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates alpha-adducin and thereby enhances the F-actin-binding activity of alpha-adducin in vitro. Here we identified the sites of phosphorylation of alpha-adducin by Rho-kinase as Thr445 and Thr480. We prepared antibody that specifically recognized alpha-adducin phosphorylated at Thr445, and found by use of this antibody that Rho-kinase phosphorylated alpha-adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated alpha-adducin accumulated in the membrane ruffling area of Madin-Darby canine kidney (MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF). The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-kinase, or alpha-adducinT445A,T480A (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migration in NRK49F cells. alpha-AdducinT445D,T480D (substitution of Thr445 and Thr480 by Asp), but not alpha-adducinT445A,T480A, counteracted the inhibitory effect of the dominant negative Rho-kinase on the TPA-induced membrane ruffling in MDCK cells. Taken together, these results indicate that Rho-kinase phosphorylates alpha-adducin downstream of Rho in vivo, and that the phosphorylation of adducin by Rho-kinase plays a crucial role in the regulation of membrane ruffling and cell motility.
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PMID:Phosphorylation of adducin by Rho-kinase plays a crucial role in cell motility. 1020 29

The human tumor suppressor Fhit protein exhibits diadenosine triphosphatase activity, hydrolyzing Ap(3)A to AMP and ADP. We report that Fhit protein efficiently cleaves the fluorogenic Ap(3)A analog diethenoadenosine triphosphate giving support to establish a simple fluorimetric assay for quantification of Fhit enzyme. Fluorimetric assays were initially tested to demonstrate that diethyl pyrocarbonate and suramin inhibit Fhit enzyme.
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PMID:Fluorimetric detection of enzymatic activity associated with the human tumor suppressor Fhit protein. 1040 61

Baculovirus phosphatase (BVP) is a member of the metazoan RNA triphosphatase enzyme family that includes the RNA triphosphatase component of the mRNA capping apparatus. BVP and other metazoan RNA triphosphatases belong to a superfamily of phosphatases that act via the formation and hydrolysis of a covalent cysteinyl-phosphate intermediate. Here we demonstrate the formation of a BVP phosphoenzyme upon reaction with [gamma-(32)P]ATP and identify the linkage as a thiophosphate based on its chemical lability. We surmise that the phosphate is linked to Cys(119) of BVP because replacement of Cys(119) by alanine or serine abrogates phosphoenzyme formation and phosphohydrolase activity. The catalytic cysteine is situated within a conserved phosphate-binding loop ((118)HCTHGINRTGY(128)). We show that all of the non-aliphatic side chains of the phosphate-binding loop are functionally important, insofar as mutants H118A, H121A, N124A, R125A, T126A, and Y128A were inactive in gamma phosphate hydrolysis and the T120A mutant was 7% as active as wild-type BVP. Structure-activity relationships at the essential positions of the phosphate-binding loop were elucidated by conservative substitutions. A conserved aspartic acid (Asp(60)) invoked as a candidate general acid catalyst was dispensable for phosphohydrolase activity and phosphoenzyme formation by BVP. We propose that the low pK(a) of the bridging oxygen of the beta phosphate leaving group circumvents a requirement for expulsion by a proton donor during attack by cysteine on the gamma phosphorus. In contrast, a conserved aspartic acid is essential for the phosphomonoesterase reactions catalyzed by protein phosphatases, where the serine or tyrosine leaving groups have a much higher pK(a) than does ADP.
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PMID:Mechanism of phosphoanhydride cleavage by baculovirus phosphatase. 1095 17

RNA triphosphatase catalyzes the first step in mRNA cap formation which entails the cleavage of the beta-gamma phosphoanhydride bond of triphosphate-terminated RNA to yield a diphosphate end that is then capped with GMP by RNA guanylyltransferase. Here we characterize a 303 amino acid RNA triphosphatase (Pct1p) encoded by the fission yeast SCHIZOSACCHAROMYCES: pombe. Pct1p hydrolyzes the gamma phosphate of triphosphate-terminated poly(A) in the presence of magnesium. Pct1p also hydrolyzes ATP to ADP and P(i) in the presence of manganese or cobalt (K(m) = 19 microM ATP; k(cat) = 67 s(-1)). Hydrolysis of 1 mM ATP is inhibited with increasing potency by inorganic phosphate (I(0.5) = 1 mM), pyrophosphate (I(0.5) = 0.4 mM) and tripolyphosphate (I(0.5) = 30 microM). Velocity sedimentation indicates that Pct1p is a homodimer. Pct1p is biochemically and structurally similar to the catalytic domain of Saccharomyces cerevisiae RNA triphosphatase Cet1p. Mechanistic conservation between Pct1p and Cet1p is underscored by a mutational analysis of the putative metal-binding site of Pct1p. Pct1p is functional in vivo in S.cerevisiae in lieu of Cet1p, provided that it is coexpressed with the S.pombe guanylyltransferase. Pct1p and other yeast RNA triphosphatases are completely unrelated, mechanistically and structurally, to the metazoan RNA triphosphatases, suggesting an abrupt evolutionary divergence of the capping apparatus during the transition from fungal to metazoan species.
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PMID:Characterization of Schizosaccharomyces pombe RNA triphosphatase. 1113 8

The bifunctional mammalian mRNA capping enzyme (Mce1) consists of an N-terminal triphosphatase domain Mce1(1-210) fused to a C-terminal guanylyltransferase domain Mce1(211-597). The physical domain order H(2)N-triphosphatase-guanylyltransferase-COOH mimics the temporal order of the capping reactions. To determine if the physical domain order is functionally important in vivo, we engineered an "inverted" mammalian capping enzyme InvMce1 [H(2)N-Mce1(211-597)-(1-210)-COOH]. We found that InvMce1 complemented the growth of Saccharomyces cerevisiae cet1delta and ceg1delta strains in which the endogenous yeast triphosphatase and guanylyltransferase genes were deleted. By testing truncated versions of InvMce1, we determined that Mce1(1-178) comprises a minimal functional triphosphatase domain. Baculovirus phosphatase (BVP) is a monofunctional single-domain protein with RNA triphosphatase and RNA diphosphatase activities and an undefined role in viral RNA metabolism. Here we demonstrated that BVP can function as an RNA triphosphatase for cap formation in vivo when fused to the C-terminus of Mce1(211-597). By characterizing a series of InvMce1-BVP derivatives with amino acid substitutions in the phosphate-binding loop of BVP, we showed that the in vivo activity of the mutant chimeras in cap formation is contingent upon in vitro phosphohydrolase activity of the respective BVP proteins. BVP catalysis in vitro was not limited to 5'-phosphorylated RNA or nucleotide substrates, but also embraced tripolyphosphatase and pyrophosphatase activities. BVP-specific activities with nucleotide and inorganic substrates were as follows: ATP (14 min(-1)), ADP (31 min(-1)), PPP(i) (3.7 min(-1)), and PP(i) (1 min(-1)). BVP did not hydrolyze AMP. We surmise that BVP has adapted the cysteinyl phosphatase fold to the hydrolysis of phosphoanhydrides.
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PMID:The domain order of mammalian capping enzyme can be inverted and baculovirus phosphatase can function in cap formation in vivo. 1250 59


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