Reformulation of an extant ATPase active site to mimic ancestral GTPase activity reveals a nucleotide base requirement for function

Hydrolysis of nucleoside triphosphates releases similar amounts of energy. However, ATP hydrolysis is typically used for energy-intensive reactions, whereas GTP hydrolysis typically functions as a switch. SpoIVA is a bacterial cytoskeletal protein that hydrolyzes ATP to polymerize irreversibly during Bacillus subtilis sporulation. SpoIVA evolved from a TRAFAC class of P-loop GTPases, but the evolutionary pressure that drove this change in nucleotide specificity is unclear. We therefore reengineered the nucleotide-binding pocket of SpoIVA to mimic its ancestral GTPase activity. SpoIVAGTPase functioned properly as a GTPase but failed to polymerize because it did not form an NDP-bound intermediate that we report is required for polymerization. Further, incubation of SpoIVAGTPase with limiting ATP did not promote efficient polymerization. This approach revealed that the nucleotide base, in addition to the energy released from hydrolysis, can be critical in specific biological functions. We also present data suggesting that increased levels of ATP relative to GTP at the end of sporulation was the evolutionary pressure that drove the change in nucleotide preference in SpoIVA.

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Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Steady state kinetic analysis of ATP hydrolysis.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)68922-2 ◽

1981 ◽

Vol 256 (16) ◽

pp. 8845-8849 ◽

Cited By ~ 8

Author(s):

G.M. Weinstock ◽

K. McEntee ◽

I.R. Lehman

Keyword(s):

Escherichia Coli ◽

Steady State ◽

Kinetic Analysis ◽

Atp Hydrolysis ◽

Reca Protein ◽

Steady State Kinetic ◽

Nucleoside Triphosphates ◽

Hydrolysis Of ◽

State Kinetic

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The reversibility of adenosine triphosphate cleavage by myosin

Biochemical Journal ◽

10.1042/bj1330323 ◽

1973 ◽

Vol 133 (2) ◽

pp. 323-328 ◽

Cited By ~ 194

Author(s):

C. R. Bagshaw ◽

D. R. Trentham

Keyword(s):

Muscle Contraction ◽

Active Site ◽

Energy Exchange ◽

Atp Hydrolysis ◽

Mechanical Energy ◽

Standard Free Energy ◽

Standard Free Energy Change ◽

Heavy Meromyosin ◽

Cleavage Step ◽

Hydrolysis Of

For the simplest kinetic model the reverse rate constants (k−1 and k−2) associated with ATP binding and cleavage on purified heavy meromyosin and heavy meromyosin subfragment 1 from rabbit skeletal muscle in the presence of 5mm-MgCl2, 50mm-KCl and 20mm-Tris–HCl buffer at pH8.0 and 22°C are: k−1<0.02s−1 and k−1=16s−1. Apparently, higher values of k−1 and k−2 are found with less-purified protein preparations. The values of k−1 and k−2 satisfy conditions required by previous 18O-incorporation studies of H218O into the Pi moiety on ATP hydrolysis and suggest that the cleavage step does involve hydrolysis of ATP or formation of an adduct between ATP and water. The equilibrium constant for the cleavage step at the myosin active site is 9. If the cycle of events during muscle contraction is described by the model proposed by Lymn & Taylor (1971), the fact that there is only a small negative standard free-energy change for the cleavage step is advantageous for efficient chemical to mechanical energy exchange during muscle contraction.

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A BIOCHEMICAL AND CYTOCHEMICAL STUDY OF ADENOSINE TRIPHOSPHATASE ACTIVITY IN THE PHLOEM OF NICOTIAN A TABACUM

The Journal of Cell Biology ◽

10.1083/jcb.60.1.221 ◽

1974 ◽

Vol 60 (1) ◽

pp. 221-235 ◽

Cited By ~ 38

Author(s):

Jamison Gilder ◽

James Cronshaw

Keyword(s):

Enzyme Activity ◽

Atpase Activity ◽

Atp Hydrolysis ◽

Phloem Transport ◽

Cytochemical Localization ◽

Gtp Hydrolysis ◽

Cytochemical Study ◽

P Protein ◽

Competitive Inhibitors ◽

Hydrolysis Of

A biochemical and cytochemical study has been made of the distribution of ATPase in mature and differentiating phloem cells of Nicotiana tabacum and of the substrate specificity and effects of fixation on enzyme activity. Homogenates of unfixed leaf midveins and midveins fixed in formaldehyde-glutaraldehyde were assayed for enzyme activity by determining the amount of Pi, liberated per milligram of protein from various substrates in a 30 min period at pH 7.2. In fresh homogenates, hydrolysis of ATP was not significantly different from that of ITP, CTP, and UTP. Hydrolysis of GTP was slightly higher than that of ATP. ATP hydrolysis by fresh homogenates was 17% more extensive than that of ADP, 76% more extensive than that of 5'-AMP, and was inhibited by fluoride and p-chloromercuribenzoate (PCMB). There was little or no hydrolysis of the competitive inhibitors 2'- and 3'-AMP nor with the alternate substrates p-nitrophenylphosphate (PNP) or ß-glycerophosphate (ß-GP). In homogenates of material fixed in formaldehyde-glutaraldehyde for 1¼ h, ATPase activity was 13% preserved. Hydrolysis of ATP by fixed homogenates was not significantly different from that of ADP, 5'-AMP, ITP, CTP, and GTP. Hydrolysis of UTP was lower. Fluoride and PCMB inhibited fixed ATPase activity. The results of cytochemical localization experiments using a lead phosphate precipitation technique were in agreement with the biochemical results. Similar localization patterns were obtained with the nucleoside triphosphates ATP, CTP, GTP, ITP, and UTP. Activity was also localized with ADP and 5'-AMP but not with the competitive inhibitors 2'- and 3'-AMP, nor with PNP or ß-GP. Little or no reaction product was deposited in other controls incubated without substrate or with substrate plus fluoride, PCMB, or N-ethylmaleimide. ATPase activity was demonstrated chiefly at the plasma membrane of mature and differentiating phloem cells and was associated with the P-protein of mature sieve elements. It is suggested that the phloem transport system derives its energy from the demonstrated nucleoside triphosphatase activity.

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Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Characterization of ATP hydrolysis.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)68920-9 ◽

1981 ◽

Vol 256 (16) ◽

pp. 8829-8834 ◽

Cited By ~ 11

Author(s):

G.M. Weinstock ◽

K. McEntee ◽

I.R. Lehman

Keyword(s):

Escherichia Coli ◽

Atp Hydrolysis ◽

Reca Protein ◽

Nucleoside Triphosphates ◽

Hydrolysis Of

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Structures of human mitofusin 1 provide insight into mitochondrial tethering

The Journal of Cell Biology ◽

10.1083/jcb.201609019 ◽

2016 ◽

Vol 215 (5) ◽

pp. 621-629 ◽

Cited By ~ 79

Author(s):

Yuanbo Qi ◽

Liming Yan ◽

Caiting Yu ◽

Xiangyang Guo ◽

Xin Zhou ◽

...

Keyword(s):

Membrane Fusion ◽

Distal Part ◽

Binding Pocket ◽

Magnesium Ion ◽

Gtpase Activity ◽

Mitochondrial Membranes ◽

Gtpase Domain ◽

Gtp Hydrolysis ◽

Mitofusin 1 ◽

Insight Into

Mitochondria undergo fusion and fission. The merging of outer mitochondrial membranes requires mitofusin (MFN), a dynamin-like GTPase. How exactly MFN mediates membrane fusion is poorly understood. Here, we determined crystal structures of a minimal GTPase domain (MGD) of human MFN1, including the predicted GTPase and the distal part of the C-terminal tail (CT). The structures revealed that a helix bundle (HB) formed by three helices extending from the GTPase and one extending from the CT closely attaches to the GTPase domain, resembling the configuration of bacterial dynamin-like protein. We show that the nucleotide-binding pocket is shallow and narrow, rendering weak hydrolysis and less dependence on magnesium ion, and that association of HB affects GTPase activity. MFN1 forms a dimer when GTP or GDP/BeF3−, but not GDP or other analogs, is added. In addition, clustering of vesicles containing membrane-anchored MGD requires continuous GTP hydrolysis. These results suggest that MFN tethers apposing membranes, likely through nucleotide-dependent dimerization.

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Inhibition of the Ecto-ATPdiphosphohydrolase of Schistosoma mansoni by Thapsigargin

Bioscience Reports ◽

10.1023/a:1010330017583 ◽

2000 ◽

Vol 20 (5) ◽

pp. 369-381 ◽

Cited By ~ 8

Author(s):

Samantha M. Martins ◽

Christiane R. Torres ◽

Sérgio T. Ferreira

Keyword(s):

Schistosoma Mansoni ◽

Active Site ◽

Calcium Transport ◽

Atp Hydrolysis ◽

External Surface ◽

Specific Inhibitor ◽

Etiologic Agent ◽

Endoplasmic Reticulum Calcium ◽

P Type ◽

Hydrolysis Of

ATPdiphosphohydrolases (ATPDases) are ubiquitous enzymes capable ofhydrolyzing nucleoside di- and triphosphates. Although a number ofpossible physiological roles have been proposed for ATPDases, detailedstudies on structure-function relationships have generally been hamperedby the lack of specific inhibitors of these enzymes. We have previouslycharacterized a Ca2+-activated ATPDase on the external surface ofthe tegument of Schistosoma mansoni, the etiologic agent of humanschistosomiasis. In the present work, we have examined the effectsof thapsigargin, a sesquiterpene lactone known as a high affinityinhibitor of sarco-endoplasmic reticulum calcium transport (SERCA)ATPase, on ATPDase activity. Whereas other lactones tested had littleor no inhibitory action, thapsigargin inhibited ATP hydrolysis by the ATPDase (Ki∼20 μM). Interestingly, hydrolysis of ADP was notinhibited by thapsigargin. The lack of inhibition of ATPase activityby orthovanadate, a specific inhibitor of P-type ATPases, and theinhibition of the Mg2+-stimulated ATP hydrolysis by thapsigarginruled out the possibility that the observed inhibition of the ATPDaseby thapsigargin could be due to the presence of contaminating SERCAATPases in our preparation. Kinetic analysis indicated that a singleactive site in the ATPDase is responsible for hydrolysis of both ATPand ADP. Thapsigargin caused changes in both Vmax and Km for ATP, indicating a mixed type of inhibition. Inhibition by thapsigarginwas little or not affected by changes in free Ca2+ or Mg2+concentrations. These results suggest that interaction of thapsigarginwith the S. mansoni ATPDase prevents binding of ATP or its hydrolysisat the active site, while ADP can still undergo catalysis.

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Structural insight into the rearrangement of the switch I region in GTP-bound G12A K-Ras

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798317015418 ◽

2017 ◽

Vol 73 (12) ◽

pp. 970-984 ◽

Cited By ~ 13

Author(s):

Shenyuan Xu ◽

Brian N. Long ◽

Gabriel H. Boris ◽

Anqi Chen ◽

Shuisong Ni ◽

...

Keyword(s):

Transition State ◽

Binding Pocket ◽

High Energy ◽

Side Chain ◽

Conformation Change ◽

Gtp Hydrolysis ◽

Structural Insight ◽

Hydrolysis Of ◽

Insight Into ◽

Intrinsic Gtpase Activity

K-Ras, a molecular switch that regulates cell growth, apoptosis and metabolism, is activated when it undergoes a conformation change upon binding GTP and is deactivated following the hydrolysis of GTP to GDP. Hydrolysis of GTP in water is accelerated by coordination to K-Ras, where GTP adopts a high-energy conformation approaching the transition state. The G12A mutation reduces intrinsic K-Ras GTP hydrolysis by an unexplained mechanism. Here, crystal structures of G12A K-Ras in complex with GDP, GTP, GTPγS and GppNHp, and of Q61A K-Ras in complex with GDP, are reported. In the G12A K-Ras–GTP complex, the switch I region undergoes a significant reorganization such that the Tyr32 side chain points towards the GTP-binding pocket and forms a hydrogen bond to the GTP γ-phosphate, effectively stabilizing GTP in its precatalytic state, increasing the activation energy required to reach the transition state and contributing to the reduced intrinsic GTPase activity of G12A K-Ras mutants.

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The Yeast Peptide Elongation Factor 3 (EF-3) Carries an Active Site for ATP Hydrolysis Which Can Interact with Various Nucleoside Triphosphates in the Absence of Ribosomes1

The Journal of Biochemistry ◽

10.1093/oxfordjournals.jbchem.a122487 ◽

1988 ◽

Vol 104 (3) ◽

pp. 445-450 ◽

Cited By ~ 17

Author(s):

Masazumi Miyazaki ◽

Masahiro Uritani ◽

Hideto Kagiyama

Keyword(s):

Active Site ◽

Atp Hydrolysis ◽

Elongation Factor ◽

Nucleoside Triphosphates ◽

Elongation Factor 3 ◽

Peptide Elongation

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Functional convergence of structurally distinct thioesterases from cyanobacteria and plants involved in phylloquinone biosynthesis

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444913015771 ◽

2013 ◽

Vol 69 (10) ◽

pp. 1876-1888 ◽

Cited By ~ 5

Author(s):

Fabienne Furt ◽

William J. Allen ◽

Joshua R. Widhalm ◽

Peter Madzelan ◽

Robert C. Rizzo ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Active Site ◽

Active Sites ◽

Binding Pocket ◽

Flowering Plant ◽

Structural Basis ◽

Photosynthetic Organisms ◽

Functional Convergence ◽

Hotdog Fold ◽

Hydrolysis Of

The synthesis of phylloquinone (vitamin K1) in photosynthetic organisms requires a thioesterase that hydrolyzes 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA) to release 1,4-dihydroxy-2-naphthoate (DHNA). Cyanobacteria and plants contain distantly related hotdog-fold thioesterases that catalyze this reaction, although the structural basis of these convergent enzymatic activities is unknown. To investigate this, the crystal structures of hotdog-fold DHNA-CoA thioesterases from the cyanobacteriumSynechocystis(Slr0204) and the flowering plantArabidopsis thaliana(AtDHNAT1) were determined. These enzymes form distinct homotetramers and use different active sites to catalyze hydrolysis of DHNA-CoA, similar to the 4-hydroxybenzoyl-CoA (4-HBA-CoA) thioesterases fromPseudomonasandArthrobacter. Like the 4-HBA-CoA thioesterases, the DHNA-CoA thioesterases contain either an active-site aspartate (Slr0204) or glutamate (AtDHNAT1) that are predicted to be catalytically important. Computational modeling of the substrate-bound forms of both enzymes indicates the residues that are likely to be involved in substrate binding and catalysis. Both enzymes are selective for DHNA-CoA as a substrate, but this selectivity is achieved using divergent predicted binding strategies. The Slr0204 binding pocket is predominantly hydrophobic and closely conforms to DHNA, while that of AtDHNAT1 is more polar and solvent-exposed. Considered in light of the related 4-HBA-CoA thioesterases, these structures indicate that hotdog-fold thioesterases using either an active-site aspartate or glutamate diverged into distinct clades prior to the evolution of strong substrate specificity in these enzymes.

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