NON-HYDRATED STATE OF THE ACYL PHOSPHATE GROUP IN THE PHOSPHORYLATED INTERMEDIATE OF (NA-ATPASE(,K+))

The position in the acyl phosphate linkage of the phosphorylated inter mediate of (Na+, K+)-ATPase that is cleaved by N-methylhydroxylamine w as compared with that of the model compound acetylphosphate. The produ cts of the cleavage of the phosphoenzyme by methylhydroxylamine were t he active enzyme and a N-P compound, not the inhibited enzyme and inor ganic phosphate, This means that the bond cleaved by methylhydroxylami ne was the O-P bond, not the C-O bond, In contrast, methylhydroxylamin e did not cleave the O-P bond of acetylphosphate in solution, at pH va lues from 0.3 to 7.0, whether or not the phosphoryl group formed a com plex with magnesium, Acetylphosphate and hydroxylamine formed acetohyd roxamic acid, Therefore, the state of the acyl phosphate bond in the n ative phosphoenzyme and in acetylphosphate in solution was different, and the difference was not due to different dissociation states of the ir phosphoryl groups or the binding of magnesium to the phosphoenzyme. Molecular orbital calculations for acetylphosphate revealed that the phosphorus atom charge is more positive than the carbon atom, irrespec tive of the dissociation state of the phosphoryl group. Similarly, the overlapping electron population of the O-P bond is always smaller tha n that of the C-O bond. Thus, the electronic structure of the acyl pho sphate linkage of acetylphosphate under vacuum supports the results ob tained with the native phosphoenzyme, rather than those obtained with acetylphosphate in solution. The linkage in the active site of the pho sphorylated intermediate of (Na+,K+)-ATPase appeared to be equivalent to the non-hydrated state of the model compound acetylphosphate. The p hosphoenzyme with bound ouabain, or without a tightly bound divalent c ation was insensitive to methylhydroxylamine. The native phosphoenzyme of (Ca2+)-ATPase was not susceptible to methylhydroxylamine.