Effects of nucleotide analogs on ATP hydrolysis by P‐type ATPases. Comparison between the canine kidney (Na⁺+ K⁺) ATPase and maize root H⁺‐ATPase

Abstract

The mechanism by which chemical energy is converted into an electrochemical gradient by P‐type ATPase is not completely understood. The effects of ATP analogs on the canine kidney (Na++ K+) ATPase were compared to effects of the same analogs on the maize (Zea mays L. cv. W7551) root H+‐ATPase in order to identify probes for the ATP binding site of the maize root enzyme and to determine potential similarities of ATP hydrolysis mechanisms in these two enzymes. Six compounds able to modify the ATP binding site covalently were compared. These compounds could be classed into three distinct groups based on activity. The first group had little or no effect on catalytic activity of either enzyme and included 7‐chloro‐4‐nitrobenz‐2‐oxa‐1.3‐diazole. The second group, which included azido adenine analogs. fluorescein isothiocyanate and 5′‐p‐fluorosulfonylbenzoyladenine, were inhibitors of ATP hydrolysis by both enzymes. However, the sensitivity of the (Na++ K+) ATPase to inhibition was much greater than that exhibited by the maize root enzyme. The third group, which included periodate treated nucleotide derivatives and 2′,3′‐o‐(4‐benzoylbenzoyl)adenosine triphosphate. inhibited both enzymes similarly. This initial screening of these covalent modifiers indicated that 2′,3′‐o‐(4‐benzoylbenzoyl)adenosine triphosphate was the optimal covalent modifier of the ATP binding site of the maize root enzyme. Certain reagents were much more effective against the (Na++ K+) ATPase than the maize root enzyme, possibly indicating differences in the ATP binding and hydrolysis pathway for these two enzymes. Two ATP analogs that are not covalent modifiers were also tested: the trinitrophenyl derivatives of adenine nucleotides were better than 5′‐adenylylimidodiphosphate for use as an ATP binding probe.

C-terminal deletion analysis of plant plasma membrane H(+)-ATPase: yeast as a model system for solute transport across the plant plasma membrane.

“…However, in these kinds of experiments, there is no way to check for the amount of active versus inactive or misfolded enzyme. An approach for future studies would be to determine the amounts of accessible, correctly folded ATP-binding sites by, for example, affini ty labeling wi t h radiolabeled 5'-(p-fluorosu Ifonyl) benzoyladenosine (Brauer and Tu, 1995).…”