scholarly journals ATPase activity associated with the magnesium-protoporphyrin IX chelatase enzyme of Synechocystis PCC6803: evidence for ATP hydrolysis during Mg2+ insertion, and the MgATP-dependent interaction of the ChlI and ChlD subunits

1999 ◽  
Vol 339 (1) ◽  
pp. 127-134 ◽  
Author(s):  
Poul E. JENSEN ◽  
Lucien C. D. GIBSON ◽  
C. Neil HUNTER
Keyword(s):  
Atpase Activity ◽  
Maximum Rate ◽  
Atp Hydrolysis ◽  
Protoporphyrin Ix ◽  
Synechocystis Pcc6803 ◽  
Absolute Requirement ◽  
Subsequent Step ◽  
A Minor ◽  
Continuous Assay ◽  
Mg Chelatase

Insertion of Mg2+ into protoporphyrin IX catalysed by the three-subunit enzyme magnesium-protoporphyrin IX chelatase (Mg chelatase) is thought to be a two-step reaction, consisting of activation followed by Mg2+ chelation. The activation step requires ATP and two of the subunits, ChlI and ChlD (I and D respectively), and it has been speculated that this step results in the formation of an I–D–ATP complex. The subsequent step, in which Mg2+ is inserted into protoporphyrin, also requires ATP and the third subunit, H, in addition to ATP-activated I–D complex. In the present study, we examine the interaction of the I and D subunits of the Mg chelatase from the cyanobacterium Synechocystis PCC 6803. We demonstrate the purification of an I–D complex, and show that ATP and Mg2+ are absolute requirements for the formation of this complex, probably as MgATP. However, ATP may be replaced by the slowly hydrolysable analogue, adenosine 5´-[γ-thio]triphosphate, and, to a minor extent, by ADP and the non-hydrolysable ATP analogue, adenosine 5´-[β,γ-imido]triphosphate, all of which suggests that ATP hydrolysis is not necessary for the formation of the ChlI–ChlD complex. A sensitive continuous assay was used to detect ATPase activity during Mg2+ chelation, and it was found that the maximum rate of ATP hydrolysis coincided with the maximum rate of Mg2+ insertion. The rate of ATP hydrolysis depended on factors that determined the rate of Mg2+ chelation, such as increasing the concentration of the H subunit and the concentration of protoporphyrin. Thus ATP hydrolysis has been identified as an absolute requirement for the chelation step. The I subunit possessed strong ATPase activity when assayed on its own, whereas the D subunit had no detectable activity, and when the I and D subunits were assayed in combination, the ATPase activity of the I subunit was repressed.

scholarly journals Modification of cysteine residues in the ChlI and ChlH subunits of magnesium chelatase results in enzyme inactivation

2000 ◽  
Vol 352 (2) ◽  
pp. 435-441 ◽  
Author(s):  
Poul E. JENSEN ◽  
James D. REID ◽  
C. Neil HUNTER
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Chlorophyll Biosynthesis ◽  
Protoporphyrin Ix ◽  
Cysteine Residue ◽  
Enzyme Inactivation ◽  
Cysteine Residues ◽  
Magnesium Chelatase ◽  
Synechocystis Pcc6803 ◽  
Chelation Reaction

The enzyme magnesium protoporphyrin chelatase catalyses the insertion of magnesium into protoporphyrin, the first committed step in chlorophyll biosynthesis. Magnesium chelatase from the cyanobacterium Synechocystis PCC6803 has been reconstituted in a highly active state as a result of purifying the constituent proteins from strains of Escherichia coli that overproduce the ChlH, ChlI and ChlD subunits. These individual subunits were analysed for their sensitivity to N-ethylmaleimide (NEM), in order to assess the roles that cysteine residues play in the partial reactions that comprise the catalytic cycle of Mg2+ chelatase, such as the ATPase activity of ChlI, and the formation of ChlI–ChlD–MgATP and ChlH–protoporphyrin complexes. It was shown that NEM binds to ChlI and inhibits the ATPase activity of this subunit, and that prior incubation with MgATP affords protection against inhibition. Quantitative analysis of the effects of NEM binding on ChlI-catalysed ATPase activity showed that three out of four thiols per ChlI molecule are available to react with NEM, but only one cysteine residue per ChlI subunit is essential for ATPase activity. In contrast, the cysteines in ChlD are not essential for Mg2+ chelatase activity, and the formation of the ChlI–ChlD–ATP complex can proceed with NEM-treated ChlI. Neither the ATPase activity of ChlI nor NEM-modifiable cysteines are therefore required to form the ChlI–ChlD–MgATP complex. However, this complex cannot catalyse magnesium chelation in the presence of the ChlH subunit, protoporphyrin and Mg2+ ions. The simplest explanation for this is that in an intact Mg2+ chelatase complex the ATPase activity of ChlI drives the chelation process. NEM binds to ChlH and inhibits the chelation reaction, and this effect can be partially alleviated by pre-incubating ChlH with magnesium and ATP. We conclude that cysteine residues play an important role in the chelation reaction, in respect of the ChlI–MgATP association, ATP hydrolysis and in the interaction of ChlH with MgATP and protoporphyrin IX.

scholarly journals Conserved L464 in p97 D1-D2 linker is critical for p97 cofactor regulated ATPase activity

2021 ◽  
Author(s):  
Xiaoyi Zhang ◽  
Lin Gui ◽  
Shan Li ◽  
Purbasha Nandi ◽  
Rod Carlo Columbres ◽  
...  
Keyword(s):  
Amino Acid ◽  
Atpase Activity ◽  
Single Particle ◽  
Maximum Rate ◽  
Atp Hydrolysis ◽  
Full Length ◽  
Wild Type ◽  
Linker Region ◽  
Functionally Diverse ◽  
The Relationship

p97 protein is a highly conserved, abundant, functionally diverse, structurally dynamic homohexameric AAA enzyme-containing N, D1, and D2 domains. A truncated p97 protein containing the N and D1 domains and the D1-D2 linker (ND1L) exhibits 79% of wild-type (WT) ATPase activity whereas the ND1 domain alone without the linker only has 2% of WT activity. To investigate the relationship between the D1-D2 linker and the D1 domain, we produced p97 ND1L mutants and demonstrated that this 22-residue linker region is essential for D1 ATPase activity. The conserved amino acid leucine 464 (L464) is critical for regulating D1 and D2 ATPase activity by p97 cofactors p37, p47, and Npl4-Ufd1 (NU). Changing leucine to alanine, proline, or glutamate increased the maximum rate of ATP turnover (kcat) of p47-regulated ATPase activities for these mutants, but not for WT. p37 and p47 increased the kcat of the proline substituted linker, suggesting that they induced linker conformations facilitating ATP hydrolysis. NU inhibited D1 ATPase activities of WT and mutant ND1L proteins, but activated D2 ATPase activity of full-length p97. To further understand the mutant mechanism, we used single-particle cryo-EM to visualize the full-length p97L464P and revealed the conformational change of the D1-D2 linker, resulting in a movement of the helix-turn-helix motif (543-569). Taken together with the biochemical and structural results we conclude that the linker helps maintain D1 in a competent conformation and relays the communication to/from the N-domain to the D1 and D2 ATPase domains, which are ~50 Å away.

Tonoplast and plasma membrane ATPases from maize lines of high or low vigour

1992 ◽  
Vol 2 (2) ◽  
pp. 105-111 ◽  
Author(s):  
S. Sánchez-Nieto ◽  
R. Rodríguez-Sotres ◽  
P. González-Romo ◽  
I. Bernal-Lugo ◽  
M. Gavilanes-Ruíz
Keyword(s):  
Zea Mays ◽  
Plasma Membrane ◽  
Acid Phosphatase ◽  
Atpase Activity ◽  
Kinetic Analysis ◽  
Atp Hydrolysis ◽  
Membrane Atpases ◽  
Substrate Concentrations ◽  
Tonoplast Atpase ◽  
Specific Inhibitors

AbstractThe effectiveness of ATPase in germinated seed may play an important role in the vigour of germination. The activities of tonoplast and plasma membrane ATPases in two maize (Zea mays L.) lines with different vigour of germination were determined. ATP hydrolysis was measured in microsomal fractions from coleoptiles along with the responses to specific inhibitors for the plasma membrane, tonoplast and mitochondrial ATPases as well as for acid phosphatase. Nitrate-sensitive ATPase activity was 1.5–3.0 times lower in the low-vigour line than in the high-vigour line. Kinetic analysis of ATP hydrolysis at different substrate concentrations revealed the existence of two enzymes in the microsomal fractions of the two lines. The Vmax of enzyme 1 in the low-vigour line was a third of that in the high-vigour line. This enzyme was identified as the nitrate-sensitive or tonoplast ATPase on the basis of measurements of ATP hydrolysis in the presence of specific inhibitors at high (8.12mm) and low (0.77mm) ATP concentrations.

scholarly journals Identification of Rab6 as an N-ethylmaleimide-sensitive fusion protein-binding protein

2000 ◽  
Vol 352 (1) ◽  
pp. 165-173 ◽  
Author(s):  
Sang Yeul HAN ◽  
Dong Yoon PARK ◽  
Sang Dai PARK ◽  
Seung Hwan HONG
Keyword(s):  
Fusion Protein ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Polyclonal Antibodies ◽  
Binding Protein ◽  
Vesicular Transport ◽  
Brain Extract ◽  
Terminal Domain ◽  
Protein Receptor ◽  
And Function

In this study we show the interaction of N-ethylmaleimide-sensitive fusion protein (NSF) with a small GTP-binding protein, Rab6. NSF is an ATPase involved in the vesicular transport within eukaryotic cells. Using the yeast two-hybrid system, we have isolated new NSF-binding proteins from the rat lung cDNA library. One of them was Rab6, which is involved in the vesicular transport within the Golgi and trans-Golgi network as a Ras-like GTPase. We demonstrated that the N-terminal domain of NSF interacted with the C-terminal domain of Rab6, and these proteins were co-immunoprecipitated from the rat brain extract. This interaction was maintained preferentially in the presence of hydrolysable ATP. Recombinant NSF-His6 can also bind to C-terminal Rab6–glutathione S-transferase under the conditions to allow the ATP hydrolysis. Surprisingly, Rab6 stimulates the ATPase activity of NSF by approx. 2-fold as does α-soluble NSF attachment protein receptor. Anti-Rab6 polyclonal antibodies significantly inhibited the Rab6-stimulated ATPase activity of NSF. Furthermore, we found that Rab3 and Rab4 can also associate with NSF and stimulate its ATPase activity. Taken together, we propose a model in which Rab can form an ATP hydrolysis-regulated complex with NSF, and function as a signalling molecule to deliver the signal of vesicle fusion through the interaction with NSF.

scholarly journals Stimulation of the human mitochondrial transporter ABCB10 by zinc-mesoporphrin

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0238754
Author(s):  
Melissa Martinez ◽  
Gregory A. Fendley ◽  
Alexandra D. Saxberg ◽  
Maria E. Zoghbi
Keyword(s):  
Oxidative Damage ◽  
Atpase Activity ◽  
Animal Studies ◽  
Atp Hydrolysis ◽  
Aminolevulinic Acid ◽  
Negative Control ◽  
Hydrolysis Rate ◽  
Inner Mitochondrial Membrane ◽  
Experimental Conditions ◽  
Mitochondrial Transporter

Heme biosynthesis occurs through a series of reactions that take place within the cytoplasm and mitochondria, so intermediates need to move across these cellular compartments. However, the specific membrane transport mechanisms involved in the process are not yet identified. The ATP-binding cassette protein ABCB10 is essential for normal heme production, as knocking down this transporter in mice is embryonically lethal and accompanied by severe anemia plus oxidative damage. The role of ABCB10 is unknown, but given its location in the inner mitochondrial membrane, it has been proposed as a candidate to export either an early heme precursor or heme. Alternatively, ABCB10 might transport a molecule important for protection against oxidative damage. To help discern between these possibilities, we decided to study the effect of heme analogs, precursors, and antioxidant peptides on purified human ABCB10. Since substrate binding increases the ATP hydrolysis rate of ABC transporters, we have determined the ability of these molecules to activate purified ABCB10 reconstituted in lipid nanodiscs using ATPase measurements. Under our experimental conditions, we found that the only heme analog increasing ABCB10 ATPase activity was Zinc-mesoporphyrin. This activation of almost seventy percent was specific for ABCB10, as the ATPase activity of a negative control bacterial ABC transporter was not affected. The activation was also observed in cysteine-less ABCB10, suggesting that Zinc-mesoporphyrin’s effect did not require binding to typical heme regulatory motifs. Furthermore, our data indicate that ABCB10 was not directly activated by neither the early heme precursor delta-aminolevulinic acid nor glutathione, downsizing their relevance as putative substrates for this transporter. Although additional studies are needed to determine the physiological substrate of ABCB10, our findings reveal Zinc-mesoporphyrin as the first tool compound to directly modulate ABCB10 activity and raise the possibility that some actions of Zinc-mesoporphyrin in cellular and animal studies could be mediated by ABCB10.

scholarly journals The Mg2+-ATPase of rabbit skeletal-muscle transverse tubule is a highly glycosylated multiple-subunit enzyme

1991 ◽  
Vol 278 (2) ◽  
pp. 375-380 ◽  
Author(s):  
T L Kirley
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Rabbit Skeletal Muscle ◽  
Cross Linking ◽  
Low Concentrations ◽  
Sds Page ◽  
Molecular Masses ◽  
Relationship Of ◽  
Peptide Core

The Mg(2+)-ATPase present in rabbit skeletal-muscle transverse tubules is an integral membrane enzyme which has been solubilized and purified previously in this laboratory [Kirley (1988) J. Biol. Chem. 263, 12682-12689]. The present study indicates that, in addition to the approx. 100 kDa protein (distinct from the sarcoplasmic-reticulum Ca(2+)-ATPase) seen previously to co-purify with the Mg(2+)-ATPase activity, there are also proteins having molecular masses of 160, 70 and 43 kDa. The 70 and 43 kDa glycosylated proteins (50 and 31 kDa after deglycosylation) are difficult to detect by SDS/PAGE before deglycosylation, owing to the broadness of the bands. Additional purification procedures, cross-linking studies and chemical and enzymic deglycosylation studies were undertaken to determine the structure and relationship of these proteins. Both the 97 and 160 kDa proteins were demonstrated to be N-glycosylated at multiple sites, the 97 kDa protein being reduced to a peptide core of 84 kDa and the 160 kDa protein to a peptide core of 131 kDa after deglycosylation. Although the Mg(2+)-ATPase activity is resistant to a number of chemical modification reagents, cross-linking inactivates the enzyme at low concentrations. This inactivation is accompanied by cross-linking of two 97 kDa molecules to one another, suggesting that the 97 kDa protein is involved in ATP hydrolysis. The existence of several proteins along with the inhibition of ATPase activity by cross-linking is consistent with the interpretation of the susceptibility of this enzyme to inactivation by most detergents as being due to the disruption of a protein complex of associated subunits by the inactivating detergents. The 160 kDa glycoprotein can be partially resolved from the Mg(2+)-ATPase activity, and is identified by its N-terminal amino acid sequence as angiotensin-converting enzyme.

scholarly journals Conformational communication mediates the reset step in t6A biosynthesis

2019 ◽  
Vol 47 (12) ◽  
pp. 6551-6567 ◽  
Author(s):  
Amit Luthra ◽  
Naduni Paranagama ◽  
William Swinehart ◽  
Susan Bayooz ◽  
Phuc Phan ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Docking ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Translational Fidelity ◽  
Substrate Analog ◽  
Binding Cavity ◽  
Saxs Analysis

Abstract The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC–AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB2D2E2) bound to Mg2+-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t6A cycle. Further, SAXS analysis of the TmTsaB2D2-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC–AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system.

Tropomyosin-Troponin-Induced Changes in the Partitioning of Free Energy Release of Actomyosin-Catalyzed ATP Hydrolysis as Measured by ATP-Phosphate Exchange

1980 ◽  
Vol 35 (5-6) ◽  
pp. 431-438 ◽  
Author(s):  
Peter Dancker
Keyword(s):  
Free Energy ◽  
Atpase Activity ◽  
Energy Release ◽  
Atp Hydrolysis ◽  
Free Energy Change ◽  
Energy Change ◽  
Experimental Conditions ◽  
Mean Values ◽  
Induced Changes ◽  
Phosphate Exchange

Abstract ATPase activity and ATP-Pi exchange of unregulated (without tropomyosin-troponin) and regulated (with tropomyosin-troponin) acto-HMM were measured in media containing 0.2 mg/ml actin, HMM, and (when present) tropomyosin-troponin, 2 mM MgCl2, 10 m M KCl, 2 mM NaN3, 10 mM Pi(pH 7.0), 3 mM ATP. The following mean values for ATPase activity and for the rate of incorporation of P, into ATP (each per mg HMM and per min) were obtained: unregulated acto-HMM 0.33 nmol Pi and 0.33 nmol Pi, regulated acto-HMM 0.54 nmol Pi and 1.06 nmol P*. The ratio of P4 incorporation rate to ATPase activity was 1.01 × 10-3 for unregulated and 2.02 × 10-3 for regulated acto-HMM. From these ratios and from the overall free energy change of ATP hydrolysis it was calculated that under the prevailing experimental conditions in unregulated acto-HMM 62% and in regulated acto-HMM 66% of the free energy change of ATP hydrolysis occurs after the release of phosphate from actomyosin. It is probably this part of the free energy change that is used by the muscle for the performance of work.

Anion-stimulated ATPase activity of brush border from rat small intestine.

1979 ◽  
Vol 236 (1) ◽  
pp. E70 ◽  
Author(s):  
M H Humphreys ◽  
L Y Chou
Keyword(s):  
Alkaline Phosphatase ◽  
Atpase Activity ◽  
Brush Border ◽  
Atp Hydrolysis ◽  
Intestinal Transport ◽  
Mitochondrial Enzymes ◽  
Intestinal Alkaline Phosphatase ◽  
Ph Optimum ◽  
Low Concentrations ◽  
Small Intestinal

Differential centrifugation of rat small intestinal homogenates produced a crude brush border (BB) fraction that was enriched 15-fold for the marker enzymes, alkaline phosphatase and sucrase; contamination with mitochondrial enzymes, monoamine oxidase and succinate dehydrogenase, was minimal. ATP hydrolysis by this BB fraction was stimulated by addition of several anions to the incubation medium: HCO3 and Cl were equally effective in this regard, with NO3, NO2, SO4, and acetate being less stimulatory. SCN and CNO inhibited ATPase activity, whereas the divalent anion SO3 was stimulatory at low concentrations (less than 25 mM) but inhibitory at 100 mM. Maximum anion stimulation was observed at a Mg concentration of 0.5 mM, and pH optimum was 8.5. Kinetic analysis showed that HCO3 increased the Vmax without altering the Km for ATP; the Ka for this effect of HCO3 was 35 mM. This enzyme activity was completely inhibited by 20 mM L-phenylalanine, 10 mM L-cysteine, and 3 mM EDTA, compounds that also inhibited intestinal alkaline phosphatase. These results demonstrate the presence of anion-stimulated ATPase activity in rat small intestinal brush border and suggest that this activity may be related to intestinal alkaline phosphatase. The role of this enzyme in intestinal transport is not known, but could relate to the regulation of intestinal absorption and secretion.

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