On the role of local unfolding in catalysis modulation

MA. PLACENTI; S. MARTINEZ GACHE; M BÁEZ LARACH; RM GONZALEZ LEBRERO; FL. GONZÁLEZ FLECHA; EA. ROMAN

Cordoba

Congreso; LI Reunión Anual de la Sociedad Argentina de Biofísica; 2023

P-ATPases constitute a family of membrane proteins that couple ATP hydrolysis to the transport of solutes across biological membranes, forming a phosphorylated intermediate during their reaction cycle. These proteins share a transmembrane domain through which the substrate is transported. Also share cytoplasmic domains that consists of a catalytic modulator domain (A or "actuator") and an ATP binding and hydrolysis domain (ATPBD or “ATP binding domain") comprised by the N ("nucleotide binding") and P ("phosphorylation") subdomains. ATPBD crystallographic structures reveal different conformational states related to open-closed movements which may be involved in coupling nucleotide hydrolysis to substrate transport. In our laboratory, we purified the ATPBD of a Cu+ transporting P-ATPase from the hyperthermophile archaea Archaeoglobus fulgidus. We characterized the effect of substrate concentration and temperature on its steady state ATPase activity observing it could be described by a Michaelis-Menten equation and obtaining Km and Vmax as a function of temperature. Additionally, we examined the effect of temperature on its intrinsic fluorescence observing a conformational transition occurring within the temperature range where this protein is catalytically active. A simple 3 states steady-state kinetic model explains the activity dependence on substrate concentration, its optimal working temperature and intrinsic fluorescence changes. Furthermore, since we hypothesized that there is an open-close transition involved in catalysis, we studied the role of local unfolding (“cracking”) on ATPBD activity and found that low urea concentrations induce an increase in ATPase activity and shift the optimal working temperature to lower values. Altogether these results suggest that, in this case, optimal working temperature is, at least partially, modulated by a conformational change.AcknowldegmentsUBA, CONICET and FONCyT