The hydrolysis of ATP by the catalytic domain of the thermophilic Cu(I) transport ATPase from Archaeoglobus fulgidus.

LURDES SABECKIS; SANTIAGO MARTINEZ; ERNESTO A. ROMAN; MARIANO C. GONZÁLEZ LEBRERO; F. LUIS GONZÁLEZ FLECHA

Salto

Congreso; Latin American Crosstalk in Biophysics and Physiology; 2015

Seccional Biofisica Sociedad Uruguaya de Biociencias

p { margin-bottom: 0.25cm; line-height: 120%; text-align: left; widows: 2; orphans: 2; }p.western { font-family: "Calibri",serif; font-size: 11pt; }p.cjk { font-size: 11pt; }Thethermophilic Cu(I) transport P-type ATPase from Archaeoglobusfulgidus(Af-CopA)is an integral membrane protein that couples the energy from ATPhydrolysis to the translocation of Cu+across cellular membranes. ATP hydrolysis takes place within thecatalytic domain (ATPBD) of AfCopAwhich is composed by two subdomains: a nucleotide binding (N-domain)and a phosphorylation (P-domain)1.The aim of this work is to study and characterize the hydrolysis ofATP within the ATPBD using a combined experimental and computationalapproach. For this purpose the ATPBD gene was cloned and its sequencewas verified. Then, it was heterologously expressed in E.coliand purified with a Ni-NTA resin. ATPase activity was measured usinga modification of the green malachite method2.The isolated ATPBD displayed optimal ATPase activity at 70ºC and pH5,5. The reaction rate was measured as a function of ATP and Mg2+concentrations obtaining a kcatvalue of 0,024 s-1with Km(ATP)=0,54 mM being Mg2+an essential activator. For computational dynamics, classical andhybrid QM/MM (quantum Mechanics-Molecular Mechanics) simulationstechniques were performed using the AMBER simulation set of programs,and a proper developed code called LIO3for the calculus of the quantum subsystem. The methyl diphosphate wasparameterized with Gaussian09 package using standard AMBER protocols.We started with the simulation of a minimal system which was thehydrolysis of methyl diphosphate. We performed classical simulationsfor the equilibration of the solvent and QM simulation for the freeenergy calculations. To obtain the free energy profile of thereaction we employed umbrella sampling calculations. The obtainedresults provide us of detailed information about the reaction of thehydrolysis of ATP by the ATPBD, which would give us valuableinformation to understand the general mechanism of catalysis of theseenzymes.References:[1]Sazinsky MH, et al. JBiol Chem.16:11161-6, 2006[2]Martinez S, González Flecha FL, en preparación 2015[3]Nitsche MA, et al,J Chem. Theory Comput,10, 959-967, 2014