On the role of local unfolding in catalysis modulation

MAURICIO BAEZ LARACH; ERNESTO A. ROMAN; SANTIAGO MARTINEZ GACHE; FRANCISCO LUIS GONZALEZ FLECHA; AGUEDA PLACENTI; RODOLFO M. GONZALEZ LEBRERO

Rosario

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

P-ATPases constitute a family of membrane proteins that couple ATP hydrolysis to thetransport of solutes across biological membranes, forming a phosphorylated intermediateduring their reaction cycle. These proteins share a transmembrane domain through whichthe substrate is transported. Also share cytoplasmic domains that consists of a catalyticmodulator 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 differentconformational states related to open-closed movements which may be involved incoupling nucleotide hydrolysis to substrate transport.In our laboratory, we purified the ATPBD of a Cu+ transporting P-ATPase from thehyperthermophile archaea Archaeoglobus fulgidus. We characterized the effect ofsubstrate concentration and temperature on its steady state ATPase activity observing itcould be described by a Michaelis-Menten equation and obtaining Km and Vmax as afunction of temperature. Additionally, we examined the effect of temperature on itsintrinsic fluorescence observing a conformational transition occurring within thetemperature range where this protein is catalytically active. A simple 3 states steady-statekinetic model explains the activity dependence on substrate concentration, its optimalworking temperature and intrinsic fluorescence changes. Furthermore, since wehypothesized that there is an open-close transition involved in catalysis, we studied therole of local unfolding (?cracking?) on ATPBD activity and found that low ureaconcentrations induce an increase in ATPase activity and shift the optimal workingtemperature to lower values. Altogether these results suggest that, in this case, optimalworking temperature is, at least partially, modulated by a conformational change.