Free energy barriers for catalysis in the ATP binding domain of the thermophilic Cu(I) transport ATPase from Archaeoglobus fulgidus.

LURDES SABECKIS; MARIANO CAMILO GONZALEZ LEBRERO; ERNESTO A. ROMAN; FRANCISCO LUIS GONZALEZ FLECHA

San Miguel de Tucuman

Congreso; Sociedad Argentina de Biofisica - LaFEBs; 2016

Sociedad Argentina de Biofisica

p { margin-bottom: 0.25cm; line-height: 120%; }The thermophilicCu(I) transport ATPase from Archaeoglobus fulgidus (Af-CopA)is an important membrane protein that couples the energy from ATPhydrolysis to the translocation of Cu(I) across plasma membranes. TheATP hydrolysis takes place within the globular domain of CopA(AfCopA-ATPBD)which is located in the intracellular portion of the cell. Itis composed by two subdomains: a nucleotide binding or N-domain and aphosphorylation or P -domain1.The product of the first reaction step is the enzime phosphorylatedat Asp 424.The aim of this workis to study and characterize the hydrolisisof ATP catalyzed by the AfCopA-ATPBDusing a combined experimental and computational approach. For this purpose theATPBD was cloned, expressed in E.Coli and purified by Ni-NTAchromatography. The ATPase activity was measured (at [Mg2+]free= 27 mM and pH=5,5) as a function of ATP concentration determiningkcatand kcat/kMat different temperatures. By fitting a Kramerstype model to the kineticcoefficients, it was obtained ∆G#,∆H#and ∆S#,following the analysisdescribed in previous reports3.In addition,computational analysiswas performed using AMBER anda self-developed code named LIO2to optimize the selected quantum subsystem calculus. Crystalstructure of Af-ATPBD(PDB 3A1C) changing AMPPCP for ATPwas used as initial structure. Itwas solvated with TIP3P water box,energy-minimized and equilibrated with classical molecular dynamicssimulation protocol.To obtain a possible reaction coordinate for the phosphorilationreaction, free energy calculations were performed using Hybrid QM/MMmethods. ΔG#values for each coordinate wereobtained using Jarzynski´s equality.Comparison betweenexperimental and theorical kinetic parametersallow us to obtainvaluable information to better understand the catalytical mechanismof Af-ATPBD and to compare it with that corresponding to the completemembrane protein3.References[1]Sazinsky MH, et al, JBiol Chem.16:11161-11166, 2006[2]Nitsche MA, et al,J Chem. Theory Comput,10:959-967, 2014[3]Bredeston, LM,GonzálezFlecha, FL, Biochim.Biophys. Acta,1858:1471-1478, 2016.p { margin-bottom: 0.25cm; line-height: 120%; }