F. Cornelius, Rate determination in phosphorylation of shark rectal Na,K-ATPase by ATP: Temperature sensitivity and effects of ADP, BIOPHYS J, 77(2), 1999, pp. 934-942
Phosphorylation of shark rectal Na,K-ATPase by ATP in the presence of Nat w
as characterized by chemical quench experiments and by stopped-flow RH421 f
luorescence. The appearance of acid-stable phosphoenzyme was faster than th
e rate of fluorescence increase, suggesting that of the two acid-stable pho
sphoenzymes formed, RH421 exclusively detects formation of E-2-P, which fol
lows formation of E-1-P. The stopped-flow RH421 fluorescence response to AT
P phosphorylation was biphasic, with a major fast phase with k(obs) similar
to 90 s(-1) and a minor slow phase with a k(obs) of similar to 9 s(-1) (20
degrees C, pH 7.4). The observed rate constants for both the slow and the
fast phase could be fitted with identical second-degree functions of the AT
P concentration with apparent binding constants of similar to 3.1 x 10(7) M
-1 and 1.8 x 10(5) M-1, respectively. Increasing [ADP] decreased k(obs) for
the rate of the RH421 fluorescence response to ATP phosphorylation. This c
ould be accounted for by the reaction of ADP with the initially formed E1P
followed by a conformational change to E-2-P. The biphasic stopped-flow RH4
21 responses to ATP phosphorylation could be simulated, assuming that in th
e absence of K+ the highly fluorescent E-2-P is slowly transformed into the
"K+-insensitive" E-2-P subconformation forming a side branch of the main c
ycle.