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)

Kar3 is a minus-end directed microtubule motor involved in meiosis and mitosis in Saccharomyces cerevisae. Unlike Drosophila Ncd, the other well characterized minus-end directed motor that is a homodimer, Kar3 is a heterodimer with a single motor domain and either the associated polypeptides Cik1 or Vik1. Our mechanistic studies with Ncd showed that both motor domains were required for ATP-dependent motor domain detachment from the microtubule. We have initiated a series of experiments to compare the mechanistic requirements for Kar3 motility in direct comparison to Ncd. The results presented here show that the single motor domain of Kar3 (Met(383)-Lys(729)) exhibits characteristics similar to monomeric Ncd. The microtubule-activated steady-state ATPase cycle of Kar3 (k(cat) = 0.5 s(-1)) is limited by ADP release (0.4 s(-1)). Like monomeric Ncd, Kar3 does not readily detach from the microtubule with the addition of MgATP. These results show that the single motor domain of Kar3 is not sufficient for ATP-dependent microtubule dissociation, suggesting that structural elements outside of the catalytic core are required for the cyclic interactions with the microtubule for force generation.
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PMID:The ATPase cross-bridge cycle of the Kar3 motor domain. Implications for single head motility. 1244 97

Menkes protein (ATP7A) is a P-type ATPase involved in copper uptake and homeostasis. Disturbed copper homeostasis occurs in patients with Menkes disease, an X-linked disorder characterized by mental retardation, neurodegeneration, connective tissue disorders, and early childhood death. Mutations in ATP7A result in malfunction of copper-requiring enzymes, such as tyrosinase and copper/zinc superoxide dismutase. The first step of the two-step amidation reaction carried out by peptidylglycine alpha-amidating monooxygenase (PAM) also requires copper. We used tissue from wild-type rats and mice and an ATP7A-specific antibody to determine that ATP7A is expressed at high levels in tissues expressing high levels of PAM. ATP7A is largely localized to the trans Golgi network in pituitary endocrine cells. The Atp7a mouse, bearing a mutation in the Atp7a gene, is an excellent model system for examining the consequences of ATP7A malfunction. Despite normal levels of PAM protein, levels of several amidated peptides were reduced in pituitary and brain extracts of Atp7a mice, demonstrating that PAM function is compromised when ATP7A is inactive. Based on these results, we conclude that a reduction in the ability of PAM to produce bioactive end-products involved in neuronal growth and development could contribute to many of the biological effects associated with Menkes disease.
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PMID:Menkes protein contributes to the function of peptidylglycine alpha-amidating monooxygenase. 1248 45

DNA mismatch repair is an essential safeguard of genomic integrity by removing base mispairings that may arise from DNA polymerase errors or from homologous recombination between DNA strands. In Escherichia coli, the MutS enzyme recognizes mismatches and initiates repair. MutS has an intrinsic ATPase activity crucial for its function, but which is poorly understood. We show here that within the MutS homodimer, the two chemically identical ATPase sites have different affinities for ADP, and the two sites alternate in ATP hydrolysis. A single residue, Arg697, located at the interface of the two ATPase domains, controls the asymmetry. When mutated, the asymmetry is lost and mismatch repair in vivo is impaired. We propose that asymmetry of the ATPase domains is an essential feature of mismatch repair that controls the timing of the different steps in the repair cascade.
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PMID:The alternating ATPase domains of MutS control DNA mismatch repair. 1255 74

The ATPase activity of MinD is required for it to oscillate between the ends of the cell and spatially regulate cell division in Escherichia coli. It is a member of a functionally diverse subgroup of ATPases which are involved in activities ranging from nitrogen fixation (NifH) to plasmid segregation (ParA). All members of the subgroup have a deviant Walker A motif which contains a conserved 'signature' lysine that characterizes this subgroup. In the NifH homodimer the signature lysines make intermonomer contact with the bound nucleotides indicating a role in ATP hydrolysis. ATP binding to NifH leads to formation of an active dimer that associates with a partner that is also a dimer. Because ATP hydrolysis is coupled to formation of the complex, the complex is only transient. In the presence of ATP MinD binds MinC and goes to the membrane, however, the ATPase is not stimulated and the complex is stable. Subsequent interaction of this complex with MinE, however, leads to ATPase stimulation and release of the Min proteins from the membrane. The sequential interaction of MinD with these two proteins, which is dictated by the membrane, is critical to the oscillatory mechanism involved in spatial regulation of division.
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PMID:MinD and role of the deviant Walker A motif, dimerization and membrane binding in oscillation. 1267 92

Copper (Cu) is an essential trace element with important roles as a cofactor in many plant functions, including photosynthesis. However, free Cu ions can cause toxicity, necessitating precise Cu delivery systems. Relatively little is known about Cu transport in plant cells, and no components of the Cu transport machinery in chloroplasts have been identified previously. Cu transport into chloroplasts provides the cofactor for the stromal enzyme copper/zinc superoxide dismutase (Cu/ZnSOD) and for the thylakoid lumen protein plastocyanin, which functions in photosynthetic electron transport from the cytochrome b(6)f complex to photosystem I. Here, we characterized six Arabidopsis mutants that are defective in the PAA1 gene, which encodes a member of the metal-transporting P-type ATPase family with a functional N-terminal chloroplast transit peptide. paa1 mutants exhibited a high-chlorophyll-fluorescence phenotype as a result of an impairment of photosynthetic electron transport that could be ascribed to decreased levels of holoplastocyanin. The paa1-1 mutant had a lower chloroplast Cu content, despite having wild-type levels in leaves. The electron transport defect of paa1 mutants was evident on medium containing <1 micro M Cu, but it was suppressed by the addition of 10 micro M Cu. Chloroplastic Cu/ZnSOD activity also was reduced in paa1 mutants, suggesting that PAA1 mediates Cu transfer across the plastid envelope. Thus, PAA1 is a critical component of a Cu transport system in chloroplasts responsible for cofactor delivery to plastocyanin and Cu/ZnSOD.
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PMID:PAA1, a P-type ATPase of Arabidopsis, functions in copper transport in chloroplasts. 1278 27

Saccharomyces cerevisiae Cet1 and Schizosaccharomyces pombe Pct1 are the essential RNA triphosphatase components of the mRNA capping apparatus of budding and fission yeast, respectively. Cet1 and Pct1 share a baroque active site architecture and a homodimeric quaternary structure. The active site is located within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel) that rests on a globular core domain (the pedestal) composed of elements from both protomers of the homodimer. Earlier studies of the effects of alanine cluster mutations at the crystallographic dimer interface of Cet1 suggested that homodimerization is important for triphosphatase function in vivo, albeit not for catalysis. Here, we studied the effects of 14 single-alanine mutations on Cet1 activity and thereby pinpointed Asp280 as a critical side chain required for dimer formation. We find that disruption of the dimer interface is lethal in vivo and renders Cet1 activity thermolabile at physiological temperatures in vitro. In addition, we identify individual residues within the pedestal domain (Ile470, Leu519, Ile520, Phe523, Leu524, and Ile530) that stabilize Cet1 in vivo and in vitro. In the case of Pct1, we show that dimerization depends on the peptide segment 41VPKIEMNFLN50 located immediately prior to the start of the Pct1 catalytic domain. Deletion of this peptide converts Pct1 into a catalytically active monomer that is defective in vivo in S. pombe and hypersensitive to thermal inactivation in vitro. Our findings suggest an explanation for the conservation of quaternary structure in fungal RNA triphosphatases, whereby the delicate tunnel architecture of the active site is stabilized by the homodimeric pedestal domain.
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PMID:Homodimeric quaternary structure is required for the in vivo function and thermal stability of Saccharomyces cerevisiae and Schizosaccharomyces pombe RNA triphosphatases. 1278 46

A homodimer of b subunits constitutes the peripheral stalk linking the F1 and F0 sectors of the Escherichia coli ATP synthase. Each b subunit has a single-membrane domain. The constraints on the membrane domain have been studied by systematic mutagenesis. Replacement of a segment proximal to the cytoplasmic side of the membrane had minimal impact on F1F0 ATP synthase. However, multiple substitutions on the periplasmic side resulted in defects in assembly of the enzyme complex. These mutants had insufficient oxidative phosphorylation to support growth, and biochemical studies showed little F1F0 ATPase and no detectable ATP-driven proton pumping activity. Expression of the b(N2A,T6A,Q10A) subunit was also oxidative phosphorylation deficient, but the b(N2A,T6A,Q10A) protein was incorporated into an F1F0 complex. Single amino acid substitutions had minimal reductions in F1F0 ATP synthase function. The evidence suggests that the b subunit membrane domain has several sites of interaction contributing to assembly of F0, and that these interactions are strongest on the periplasmic side of the bilayer.
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PMID:Mutagenesis studies of the F1F0 ATP synthase b subunit membrane domain. 1474 Aug 87

The Atx1 copper metallochaperone from Synechocystis PCC 6803, ScAtx1, interacts with two P(1)-type copper ATPases to supply copper proteins within intracellular compartments, avoiding ATPases for other metals en route. Here we report NMR-derived solution structures for ScAtx1. The monomeric apo form has a betaalphabetabetaalpha fold with backbone motions largely restricted to loop 1 containing Cys-12 and Cys-15. The tumbling rate of Cu(I)ScAtx1 (0.1-0.8 mm) implies dimers. Experimental restraints are satisfied by symmetrical dimers with Cys-12 or His-61, but not Cys-15, invading the copper site of the opposing subunit. A full sequence of copper ligands from the cell surface to thylakoid compartments is proposed, considering in vitro homodimer liganding to mimic in vivo liganding in ScAtx1-ATPase heterodimers. A monomeric high resolution structure for Cu(I)ScAtx1, with Cys-12, Cys-15, and His-61 as ligands, is calculated without violations despite the rotational correlation time. (2)J(NH) couplings in the imidazole ring of His-61 establish coordination of N(epsilon2) to copper. His-61 is analogous to Lys-65 in eukaryotic metallochaperones, stabilizing Cu(I)S(2) complexes but by binding Cu(I) rather than compensating charge. Cys-Cys-His ligand sets are an emergent theme in some copper metallochaperones, although not in related Atx1, CopZ, or Hah1. Surface charge (Glu-13) close to the metal-binding site of ScAtx1 is likely to support interaction with complementary surfaces of copper-transporting ATPases (PacS-Arg-11 and CtaA-Lys-14) but to discourage interaction with zinc ATPase ZiaA and so inhibit aberrant formation of copper-ZiaA complexes.
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PMID:Solution structures of a cyanobacterial metallochaperone: insight into an atypical copper-binding motif. 1507 18

Copper, like iron, is a transition metal that can generate oxygen radicals by the Fenton reaction. The Plasmodium parasite invades an erythrocyte host cell containing 20 microM copper, of which 70% is contained in the Cu/Zn SOD (cuprozinc superoxide dismutase). In the present study, we follow the copper pathways in the Plasmodium-infected erythrocyte. Metal-determination analysis shows that the total copper content of Percoll-purified trophozoite-stage-infected erythrocytes is 66% that of uninfected erythrocytes. This decrease parallels the decrease seen in Cu/Zn SOD levels in parasite-infected erythrocytes. Neocuproine, an intracellular copper chelator, arrests parasites at the ring-to-trophozoite stage transition and also specifically decreases intraparasitic levels of Cu/Zn SOD and catalase. Up to 150 microM BCS (2,9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedisulphonic acid), an extracellular copper chelator, has no effect on parasite growth. We characterized a single copy PfCuP-ATPase (Plasmodium falciparum copper P-ATPase) transporter, which, like the Crypto-sporidium parvum copper P-ATPase, has a single copper-binding domain: 'Met-Xaa-Cys-Xaa-Xaa-Cys'. Recombinant expression of the N-terminal metal-binding domain reveals that the protein specifically binds reduced copper. Transcription of the PfCuP-ATPase gene is the highest at late ring stage/early trophozoite, and is down-regulated in the presence of neocuproine. Immunofluorescence and electron microscopy indicate the transporter to be both in the parasite and on the erythrocyte membrane. Both the decrease in total copper and the location of the PfCuP-ATPase gene indicate a copper-efflux pathway from the infected erythrocyte.
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PMID:Copper pathways in Plasmodium falciparum infected erythrocytes indicate an efflux role for the copper P-ATPase. 1512 86

Structural maintenance of chromosomes (SMC) proteins are central regulators of higher-order chromosome dynamics from bacteria to humans. The Bacillus subtilis SMC (BsSMC) homodimer adopts a V-shaped structure with an ATP-binding catalytic domain at each end. We report here that two small proteins, ScpA and ScpB, associate with the catalytic domains of BsSMC in an ordered fashion and suppress its ATPase activity. When combined with a 'transition state' mutant of BsSMC that poorly hydrolyzes ATP, ScpA promotes stable engagement of two catalytic domains in an ATP-dependent manner. In solution, this occurs intramolecularly and closes the DNA-entry gate of an SMC dimer. ScpB further stabilizes this conformation and prevents BsSMC from binding to double-stranded DNA (dsDNA). In contrast, when the mutant BsSMC is first allowed to interact with dsDNA, subsequent addition of ScpA leads to assembly of large nucleoprotein complexes, possibly by stabilizing intermolecular engagement of the catalytic domains from different SMC dimers. We propose that the ATP-modulated engagement/disengagement cycle of SMC proteins plays both positive and negative roles in their dynamic interactions with dsDNA.
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PMID:Positive and negative regulation of SMC-DNA interactions by ATP and accessory proteins. 1517 56


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