Pre-steady state kinetic studies on H+-ATPase front Candida albicans

The mechanism of ATP hydrolysis by plasma membrane H+-ATPase from Candida a lbicans has been investigated by following the kinetics of H+ liberation/ab sorption and the UV difference spectrum in a stopped flow spectrophotometer , A distinct pre-steady state phase of ATP hydrolysis could be defined. Whi le the rapid mixing of pi and ATPase produced no transient pH changes, the mixing of ADP leads to the release of 1 H+ per molecule of ATPase, Rapid mi xing of ATP with ATPase releases about 2 H+ per molecule of ATPase, of whic h around 1.3 H+ are reabsorbed. The magnitudes of both H+ release and absor ption were found to be independent of ATP concentration, The rate of H+ rel ease (kr) shows ATP dependence while the rate of Hi absorption is independe nt of ATP concentration. The rate of H+ liberation with ADP, on a concentra tion basis, was far less as compared with ATP, indicating a low affinity of the ATPase for ADP. No change in the difference spectrum was observed with ADP, The stoichiometry of ATP binding to PM-ATPase was found to be unity f rom ITV-difference spectrum studies. The k(1) values for H+ release and for the appearance of a difference spectrum following the addition of ATP were found to be similar beyond a I:1 ratio of ATP:ATPase, The results obtained lead us to propose a 4-step kinetic scheme for the mechanism of ATP hydrol ysis.