The yeast plas
ma
me
mbrane H
+-ATPase isolation procedure was i
mproved; a highly pureenzy
me (90-95%) was obtained after centrifugation on a trehalose concentration gradient. H
+-ATPasekinetics was slightly cooperative: Hill nu
mber = 1.5,
S0.5 = 800
mages/entities/
mgr.gif">M ATP, and turnover nu
mber = 36s
-1. In contrast to those of other P-type ATPases, H
+-ATPase fluorescence was highly sensitive to nucleotidebinding; the fluorescence decreased 60% in the presence of both 5
mM ADP and AMP-PNP. Fluorescencetitration with nucleotides allowed calculation of dissociation constants (
Kd) fro
m the binding site;
Kdvalues for ATP and ADP were 700 and 800
mages/entities/
mgr.gif">M, respectively. On the basis of a
mino acid sequence andho
mology
model analysis, we propose that binding of the nucleotide to the N-do
main is coupled to the
move
ment of a loop
mages/gifchars/beta2.gif" BORDER=0 ALIGN="
middle"> structure and to the exposure of the Trp505 residue located in the loop. Thereco
mbinant N-do
main also displayed a large hyperbolic fluorescence quenching when ATP binds; however,it displayed a higher affinity for ATP (
Kd = 100
mages/entities/
mgr.gif">M). We propose for P-type ATPases that structural
move
ments during nucleotide binding could be followed if a Trp residue is properly located in the N-do
main.Further, we propose the use of trehalose in enzy
me purification protocols to increase the purity and qualityof the isolated protein and to perfor
m structural studies.