共查询到20条相似文献,搜索用时 31 毫秒
1.
We characterized the activities of the Myxococcus xanthus ApaH-like phosphatases PrpA and ApaH, which share homologies with both phosphoprotein phosphatases and diadenosine tetraphosphate (Ap4A) hydrolases. PrpA exhibited a phosphatase activity towards p-nitrophenyl phosphate (pNPP), tyrosine phosphopeptide and tyrosine-phosphorylated protein, and a weak hydrolase activity towards ApnA and ATP. In the presence of Mn2+, PrpA hydrolyzed Ap4A into AMP and ATP, whereas in the presence of Co2+ PrpA hydrolyzed Ap4A into two molecules of ADP. ApaH exhibited high phosphatase activity towards pNPP, and hydrolase activity towards ApnA and ATP. Mn2+ was required for ApaH-mediated pNPP dephosphorylation and ATP hydrolysis, whereas Co2+ was required for ApnA hydrolysis. Thus, PrpA and ApaH may function mainly as a tyrosine protein phosphatase and an ApnA hydrolase, respectively. 相似文献
2.
《Phytochemistry》1986,26(1):65-69
In the presence of K+, the hydrolysis of ATP catalysed by the ATPase of corn plasma membrane showed negative cooperative kinetics. When the complexes of ATP and Mg2+, Mn2+, Ca2+ or Cd2+ were used as substrates, the catalysed hydrolysis changed to follow simple Michaelis-Menten kinetics. However, this change was not observed with Zn2+-ATP as the substrate. A substantial enhancement of the hydrolysis was observed only when the complexes of Mg2+ and Mn2+ were used. Kinetic parameter determination indicated that the enzyme exhibited a similar binding property but a different catalytic efficiency to Mg2+, Mn2+ and Ca2+-ATP. The enzyme formed a more stable but less reactive complex with Cd2+-ATP. The presence of aluminium ions competitively inhibited the membrane-catalysed hydrolysis of Mg2+-ATP, but showed no effect when free ATP was the substrate. This finding suggested that aluminium might bind in the vicinity of the Mg2+ of Mg2+-ATP in the active site of the enzyme. On the basis of these observed inhibitory effects, possible origins of metal ion toxicity to root plasma membrane ATPase activity are discussed. 相似文献
3.
The binding properties of Mg2+, Mn2+ to yeast mitochondrial oligomycin sensitive ATPase complex are studied, as reflected by their catalytic effect (hydrolysis of ATP or pNPP, a pseudo substrate) or by a physical parameter (atomic absorption, electron paramagnetic resonance of Mn2+, enhanced fluorescence of chelating chlorotetracyclin). At least two classes of sites with very different affinities respectively around 10−5 M and 10−4 M are demonstrated : high affinity sites for cations which participate in pNPP hydrolysis and can bind ADP or ATP, although they have a poor efficiency for ATP hydrolysis, and low affinity sites for cations which participate efficiently in both pNPP and ATP hydrolysis. The possibility that the tight site class has itself two sub-classes is also discussed. 相似文献
4.
Mycobacterium tuberculosis RecA intein (PI-MtuI), a LAGLIDADG homing endonuclease, displays dual target specificity in response to alternative cofactors. While both ATP and Mn2+ were required for optimal cleavage of an inteinless recA allele (hereafter referred to as cognate DNA), Mg2+ alone was sufficient for cleavage of ectopic DNA sites. In this study, we have explored the ability of PI-MtuI to catalyze ATP hydrolysis in the presence of alternative metal ion cofactors and DNA substrates. Our results indicate that PI-MtuI displays maximum ATPase activity in the presence of cognate but not ectopic DNA. Kinetic analysis revealed that Mn2+ was able to stimulate PI-MtuI catalyzed ATP hydrolysis, whereas Mg2+ failed to do so. Using UV crosslinking, limited proteolysis and amino acid sequence analysis, we show that 32P-labeled ATP was bound to a 14 kDa peptide containing the putative Walker A motif. Furthermore, the limited proteolysis approach disclosed that cognate DNA was able to induce structural changes in PI-MtuI. Mutation of the presumptive metal ion-binding ligands (Asp122 and Asp222) in the LAGLIDADG motifs of PI-MtuI impaired its affinity for ATP, thus resulting in a reduction in or loss of its endonuclease activity. Together, these results suggest that PI-MtuI is a (cognate) DNA- and Mn2+-dependent ATPase, unique from the LAGLIDADG family of homing endonucleases, and implies a possible role for ATP hydrolysis in the recognition and/or cleavage of homing site DNA sequence. 相似文献
5.
M. V. Monakhova A. I. Penkina A. V. Pavlova A. M. Lyaschuk V. V. Kucherenko A. V. Alexeevski V. G. Lunin P. Friedhoff G. Klug T. S. Oretskaya E. A. Kubareva 《Biochemistry. Biokhimii?a》2018,83(3):281-293
We have purified the MutL protein from Rhodobacter sphaeroides mismatch repair system (rsMutL) for the first time. rsMutL demonstrated endonuclease activity in vitro, as predicted by bioinformatics analysis. Based on the alignment of 1483 sequences of bacterial MutL homologs with presumed endonuclease activity, conserved functional motifs and amino acid residues in the rsMutL sequence were identified: five motifs comprising the catalytic site responsible for DNA cleavage were found in the C–terminal domain; seven conserved motifs involved in ATP binding and hydrolysis and specific to the GHKL family of ATPases were found in the N–terminal domain. rsMutL demonstrated the highest activity in the presence of Mn2+. The extent of plasmid DNA hydrolysis declined in the row Mn2+ > Co2+ > Mg2+ > Cd2+; Ni2+ and Ca2+ did not activate rsMutL. Divalent zinc ions inhibited rsMutL endonuclease activity in the presence of Mn2+ excess. ATP also suppressed plasmid DNA hydrolysis by rsMutL. Analysis of amino acid sequences and biochemical properties of five studied bacterial MutL homologs with endonuclease activity revealed that rsMutL resembles the MutL proteins from Neisseria gonorrhoeae and Pseudomonas aeruginosa. 相似文献
6.
The catalytic activity of guanylate cyclase (GCase) coupled to atrial natriuretic peptide (ANP) receptor depends on the metal co-factor, Mn2+ or Mg2+. ATP synergistically stimulates the ANP-stimulated GCase in the presence of Mg2+. We have now shown the ATP regulation of the ANP-stimulated GCase in the presence of Mn2+ in rat lung membranes. ANP stimulated the GCase 2.1-fold compared to the control. ATP enhanced both the basal (basal-GCase) and the ANP-stimulated GCase maximally 1.7- and 2.3- fold compared to the control, respectively, at a concentration of 0.1 mM. The stimulation by ATP was smaller in the presence of Mn2+ than in the presence of Mg2+. The addition of inorganic phosphate to the reaction mixture altered the GCase activities in the presence of Mn2+ with or without ANP and/or ATP. In the presence of 10 mM phosphate, ATP dose-dependently stimulated the basal GCase 5-fold compared to the control at a concentration of 1 mM and augmented the ANP-stimulated GCase, which was 4.2-fold compared to the basal-GCase, 5.5-fold compared to the control at a concentration of 0.5 mM. Protein phosphatase inhibitors, okadaic acid (100 nM), H8 (1 M) and staurosporin (1 M), did not alter the activity. Orthovanadate (1 mM), an inorganic phosphate analogue, significantly stimulated both the basal-GCase and the ANP-stimulated GCase, which were inhibited by ATP. It was assumed that phosphate and orthovanadate might interact with the GCase to regulate the activity in the opposite manner. This was the first report that inorganic phosphate and orthovanadate affected the ATP-regulation of the ANP-stimulated GCase in the presence of Mn2+. 相似文献
7.
Aspartate kinase and two homoserine dehydrogenases were partially purified from 4-day-old pea seedlings. A sensitive method for measuring aspartate kinase activity is described. Aspartate kinase activity was dependent upon ATP, Mg2+ or Mn2+, and aspartate. The aspartate kinase was inhibited in a sigmoidal manner by threonine and Ki for threonine was 0·57 mM. The enzyme could be desensitized to the inhibitor and threonine protected the enzyme against thermal inactivation. Aspartate kinase activity was enhanced by isoleucine, valine and alanine. Homoserine, methionine and lysine were without effect. The homoserine dehydrogenase activity which was associated with aspartate kinase during purification could be resolved into two peaks by gel filtration. The activity of both peaks was inhibited by aspartate and cysteine and one was inhibited by threonine. 相似文献
8.
Camila C. Ribeiro Renan M. Monteiro Flavia P. Freitas Claudio Retamal Layz R. S. Teixeira Livia M. Palma Flavia E. Silva Arnoldo R. Fa?anha Anna L. Okorokova-Fa?anha Lev A. Okorokov 《PloS one》2012,7(11)
V H+-ATPase has an important role in a variety of key physiological processes. This enzyme is reversibly activated/partly inactivated by the addition/exhaustion of extracellular glucose. The current model of its regulation assumes the reversible disassembly/reassembly of ∼60–70% of the V1 and V0 membrane complexes, which are responsible for ATP hydrolysis and H+ conductance, respectively. The number of assembled complexes determines the pump activity because disassembled complexes are inactive. The model predicts the identical catalytic properties for the activated and semi-active enzymes molecules. To verify the model predictions we have isolated total membranes from yeast spheroplasts that were pre-incubated either with or without glucose. Nitrate treatment of membranes revealed the similar ATPase inhibition for two enzyme states, suggesting that they have identical structures that are essential for ATP hydrolysis. However, H+ transport was inhibited more than the ATPase activities, indicating a nitrate uncoupling action, which was significantly higher for the nonactivated enzyme. This finding suggests that the structure of the non-activated enzyme, which is essential for H+ transport, is less stable than that of the activated enzyme. Moreover, the glucose activation of the pump increases i) its coupling capacity; ii) its KM for ATP hydrolysis and ATP affinity for H+ transport; iii) the Vmax for H+ transport in comparison with the Vmax for ATP hydrolysis and iv) the immune reactivity of catalytic subunit A and regulatory subunit B by 9.3 and 2.4 times, respectively. The protein content of subunits A and B was not changed by extracellular glucose. We propose that instead of the dissociation/reassociation of complexes V1 and V0, changes in the extracellular glucose concentration cause reversible and asymmetrical modulations in the immune reactivity of subunits A and B by their putative biochemical modifications. This response asymmetrically modulates H+-transport and ATP hydrolysis, exhibiting distinct properties for the activated versus non-activated enzymes. 相似文献
9.
Charlène Cornilleau Noureddine Atmane Eric Jacquet Callum Smits Juan C. Alonso Paulo Tavares Leonor Oliveira 《Nucleic acids research》2013,41(1):340-354
The large terminase subunit is a central component of the genome packaging motor from tailed bacteriophages and herpes viruses. This two-domain enzyme has an N-terminal ATPase activity that fuels DNA translocation during packaging and a C-terminal nuclease activity required for initiation and termination of the packaging cycle. Here, we report that bacteriophage SPP1 large terminase (gp2) is a metal-dependent nuclease whose stability and activity are strongly and preferentially enhanced by Mn2+ ions. Mutation of conserved residues that coordinate Mn2+ ions in the nuclease catalytic site affect the metal-induced gp2 stabilization and impair both gp2-specific cleavage at the packaging initiation site pac and unspecific nuclease activity. Several of these mutations block also DNA encapsidation without affecting ATP hydrolysis or gp2 C-terminus binding to the procapsid portal vertex. The data are consistent with a mechanism in which the nuclease domain bound to the portal switches between nuclease activity and a coordinated action with the ATPase domain for DNA translocation. This switch of activities of the nuclease domain is critical to achieve the viral chromosome packaging cycle. 相似文献
10.
β-aspartokinase (EC 2.7.2.4.) has been isolated from the developing endosperm (30 days post-pollination) of Zea mays (cv. Pioneer 3145). Enzyme activity was dependent upon ATP, Mg++ or Mn++, aspartate, and protein concentration. Double reciprocal plots of velocity vs. aspartate concentrations deviated from a straight line at low aspartate concentration indicating two apparent Km's of 0.5 and 6.6 mM. Enzyme activity was inhibited by lysine but not by methionine or threonine. The endosperm-derived β-aspartokinase behaved similarly to enzyme isolated from 6-day-old etiolated shoot tissue. The presence of β-aspartokinase in developing endosperm provides new insight into the source of the aspartate-derived amino acids in maize endosperm. 相似文献
11.
Jürgen Ulrich Linder 《PloS one》2015,10(10)
YHS-domains are small protein modules which have been proposed to bind transition-metal ions like the related TRASH-domains. They are found in a variety of enzymes including copper-transporting ATPases and adenylyl cyclases. Here we investigate a class IIIc adenylyl cyclase from Mycobacterium phlei which contains a C-terminal YHS-domain linked to the catalytic domain by a peptide of 8 amino acids. We expressed the isolated catalytic domain and the full-length enzyme in E. coli. The catalytic domain requires millimolar Mn2+ as a cofactor for efficient production of cAMP, is unaffected by low micromolar concentrations of Cu2+ and inhibited by concentrations higher than 10 μM. The full-length enzyme also requires Mn2+ in the absence of an activator. However, 1–10 μM Cu2+ stimulate the M. phlei adenylyl cyclase sixfold when assayed with Mn2+. With Mg2+ as the probable physiological cofactor of the adenylyl cyclase Cu2+ specifically switches the enzyme from an inactive to an active state. Other transition-metal ions do not elicit activity with Mg2+. We favor the view that the YHS-domain of M. phlei adenylyl cyclase acts as a sensor for copper ions and signals elevated levels of the transition-metal via cAMP. By analogy to TRASH-domains binding of Cu2+ probably occurs via one conserved aspartate and three conserved cysteine-residues in the YHS-domain. 相似文献
12.
The activation and catalytic mechanism of corn mitochondrial F1 were examined for the two distinct forms of the enzyme which appear upon storage in ammonium sulfate or glycerol. Apparently irreversible differences in the stability of the two active forms were found. Nucleosidetriphosphate induced activation of the enzyme was found to produce lasting effects on subsequent catalysis. These effects varied with both the nucleotide used for activation, and the hydrolyzed species. The substrate and metal specificity were examined with the ATP activated enzyme. Mg2+ and Ca2+ were found to be the most effective at promoting ATP hydrolysis. The substrates were hydrolyzed in the order GTP > ITP > ATP regardless of which nucleotide was used for activation. While ATP and GTP hydrolysis exhibited kinetics typical of other ATPases, ITP showed a transition from negative to positive cooperativity at low substrate concentrations. Bicarbonate was found to affect primarily the kinetics of ATP hydrolysis. AMP-PNP proved to be a potent inhibitor with respect to ATP hydrolysis. The results are discussed in terms of possible catalytic mechanisms and the similarities of the corn mitochondrial F1 to other ATPases. 相似文献
13.
The membrane-bound atrial natriuretic peptide receptor (GCA) catalyzes the formation of cGMP from GTP in response to natriuretic peptide hormones. Previous structural studies have focused on the extra-cellular hormone binding domain of this receptor whereas its intra-cellular domain has not yet been amenable to such studies. We report here the baculovirus expression and purification of the GCA intra-cellular domain construct GCAID comprising the complete intra-cellular region which includes the kinase-homology domain, coiled-coil region, and catalytic cyclase domain. The intra-cellular domain was enzymatically characterized in terms of guanylyl cyclase activity and the effects of ATP, manganese, and Triton X-100. Our results indicate that the activity of the intra-cellular domain of the ANP receptor is about 2 fold less active compared to a truncated cyclase domain construct lacking the kinase-like domain that was also expressed and purified. In addition, unlike the full length receptor, the intra-cellular domain could not be activated by Triton X-100/Mn2+ or its activity stimulated by ATP. These data therefore indicate that the major part of the transition from the basal state to the fully, ANP/ATP-dependent, activated state as well its stimulation/enhancement by Triton X-100/Mn2+ requires the full length receptor. These receptor insights could aid in the development of novel therapeutics as the GCA receptor is a key drug target for cardiovascular diseases. 相似文献
14.
When an ATPase Is Not an ATPase: at Low Temperatures the C-Terminal Domain of the ABC Transporter CvaB Is a GTPase
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins which share a common function and a common nucleotide-binding domain. The CvaB protein from Escherichia coli is a member of the bacterial ABC exporter subfamily and is essential for the export of the peptide antibiotic colicin V. Here we report that, surprisingly, the CvaB carboxyl-terminal nucleotide-binding domain (BCTD) can be preferentially cross-linked to GTP but not to ATP at low temperatures. The cross-linking is Mg2+ and Mn2+ dependent. However, BCTD possesses similar GTPase and ATPase activities at 37°C, with the same kinetic parameters and with similar responses to inhibitors. Moreover, a point mutation (D654H) in CvaB that completely abolishes colicin V secretion severely impairs both GTPase and ATPase activities in the corresponding BCTD, indicating that the two activities are from the same enzyme. Interestingly, hydrolysis activity of ATP is much more cold sensitive than that of GTP: BCTD possesses mainly GTP hydrolysis activity at 10°C, consistent with the cross-linking results. These findings suggest a novel mechanism for an ABC protein-mediated transport with specificity for GTP hydrolysis. 相似文献
15.
Mauricio Angel Seigelchifer Susana Passeron 《Archives of biochemistry and biophysics》1984,229(1):403-413
Soluble preparations from mycelium of the dimorphic fungus Mucor rouxii contained detectable amounts of phosphoprotein phosphatase activity. This cytosolic phosphatase activity exhibited a molecular weight below 80,000 and could be resolved into two different forms (enzymes I and II) by chromatography on DEAE-cellulose followed by gel filtration on Sephacryl S-300. Enzyme I (Mr 64,000) was mainly a histone phosphatase activity, absolutely dependent on divalent cations, with a K0.5 for MnCl2 of 2 mm. Enzyme II (Mr 40,000) was active with histone and phosphorylase. Its activity was independent or slightly inhibited by Mn2+. This enzyme was strongly inhibited by 50 mm NaF or 1 mm ATP. When partially purified enzymes I and II were separately treated with ethanol, the catalytic properties of enzyme II were apparently not affected while those of enzyme I were drastically changed. The activity with histone, which was originally dependent on Mn2+, became independent or slightly inhibited by the cation. The treatment was accompanied by a notable increase in phosphorylase phosphatase activity which was strongly inhibited by Mn2+. Treated enzyme I eluted from DEAE-cellulose and Sephacryl S-300 columns at a position similar to that of enzyme II. 相似文献
16.
Intestinal guanylate cyclase C is activated by guanylin, an endogenous peptide. This activity seems to be modulated by adenine nucleotides, the ions Mg2+ and Mn2+, and pH. In this study, we report an ultracytochemical method for the localization of guanylate cyclase C activity at the electron microscope level. We studied the enzymatic activity in the presence or absence of guanylin and/or ATP, in the presence of the ions Mg2+ or Mn2+, and at different pH levels. The greatest distribution of enzymatic activity was detected in samples incubated at pH 8 and 7.4 in the presence of guanylin, Mg2+ and ATP. Guanylate cyclase C activity was detected at the surface epithelium of stomach and intestine, and in liver, exocrine pancreas and parotid gland. In the intestine, enzymatic activity was more widely distributed in the duodenum than in the jejunum–ileum and colon. In the small intestine, activity was more evident in the upper portion than in the basal portion of the villus. In samples incubated at pH 8 and 7.4 in the absence of ATP, enzymatic activity was detected only in small intestine, liver and exocrine pancreas. Enzymatic activity was present in duodenum incubated at pH 8 and 7.4 in the presence of Mn2+ and in the presence or absence of ATP. No samples incubated in all these experimental conditions but at pH 5 or samples incubated in the presence of guanylin only or in the absence of guanylin, displayed guanylate cyclase C activity. Our results suggest that a complete ultracytochemical detection of guanylate cyclase C activity requires guanylin as stimulator, and incubation in the presence of Mg2+ and ATP atbreak pH 8 and 7.4. 相似文献
17.
The mechanism by which chemical energy is converted into an electrochemical gradient by P-type ATPase is not completely understood. The effects of ATP analogs on the canine kidney (Na++ K+) ATPase were compared to effects of the same analogs on the maize (Zea mays L. cv. W7551) root H+-ATPase in order to identify probes for the ATP binding site of the maize root enzyme and to determine potential similarities of ATP hydrolysis mechanisms in these two enzymes. Six compounds able to modify the ATP binding site covalently were compared. These compounds could be classed into three distinct groups based on activity. The first group had little or no effect on catalytic activity of either enzyme and included 7-chloro-4-nitrobenz-2-oxa-1.3-diazole. The second group, which included azido adenine analogs. fluorescein isothiocyanate and 5′-p-fluorosulfonylbenzoyladenine, were inhibitors of ATP hydrolysis by both enzymes. However, the sensitivity of the (Na++ K+) ATPase to inhibition was much greater than that exhibited by the maize root enzyme. The third group, which included periodate treated nucleotide derivatives and 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate. inhibited both enzymes similarly. This initial screening of these covalent modifiers indicated that 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate was the optimal covalent modifier of the ATP binding site of the maize root enzyme. Certain reagents were much more effective against the (Na++ K+) ATPase than the maize root enzyme, possibly indicating differences in the ATP binding and hydrolysis pathway for these two enzymes. Two ATP analogs that are not covalent modifiers were also tested: the trinitrophenyl derivatives of adenine nucleotides were better than 5′-adenylylimidodiphosphate for use as an ATP binding probe. 相似文献
18.
Oksana Gerlits Jianhui Tian Amit Das Paul Langan William T. Heller Andrey Kovalevsky 《The Journal of biological chemistry》2015,290(25):15538-15548
To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca2+ ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex, the thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca2+ cations with Mg2+ ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. The present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date. 相似文献
19.
The effect of Mn2+, a known mutagen, on the fidelity of DNA synthesis by avian myeloblastosis DNA polymerase has been determined. Substitution of Mn2+ for Mg2+ leads to an enhanced incorporation of noncomplementary deoxynucleotides as well as complementary ribonucleotides with either poly (A) or poly (C) as templates. Since this polymerase lacks any detectable deoxyribonuclease activity, the mutagenic effect of Mn2+ in promoting errors in base-pairing does not result from any diminished proof-reading function. 相似文献
20.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(4):233-241
AbstractArylsulfate sulfotransferase (ASST) from a human intestinal bacterium stoichiometrically catalyzed the transfer of a sulfate group from phenylsulfate esters to phenolic compounds. Pentachlorophenol, one of the selective inhibitors of phenol sulfoconjugation in mammalian tissues, inhibited both phenol and tyramine sulfation by ASST Nucleotide triphosphaies such as ATP, GTP, UTP and CTP, and pyrophosphate inhibited the ASST activity, whereas Mg2+ and Mn2+ activated the enzyme and prevented its inhibition by ATP and pyrophosphate. Equimolar binding of [α-] and [γ-32P]ATP to the enzyme showed that the enzyme protein was not phospholylated, but bound ATP. These results suggest that nucleotide triphosphates and divalent cations are important modulators in the control of ASST activity. 相似文献