Abstract:
:The position in the acyl phosphate linkage of the phosphorylated intermediate of (Na+, K+)-ATPase that is cleaved by N-methylhydroxylamine was compared with that of the model compound acetylphosphate. The products of the cleavage of the phosphoenzyme by methylhydroxylamine were the active enzyme and a N-P compound, not the inhibited enzyme and inorganic phosphate. This means that the bond cleaved by methylhydroxylamine was the O-P bond, not the C-O bond. In contrast, methylhydroxylamine did not cleave the O-P bond of acetylphosphate in solution, at pH values from 0.3 to 7.0, whether or not the phosphoryl group formed a complex with magnesium. Acetylphosphate and hydroxylamine formed acetohydroxamic acid. Therefore, the state of the acyl phosphate bond in the native phosphoenzyme and in acetylphosphate in solution was different, and the difference was not due to different dissociation states of their 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, irrespective of the dissociation state of the phosphoryl group. Similarly, the overlapping electron population of the O-P bond is always smaller than that of the C-O bond. Thus, the electronic structure of the acyl phosphate linkage of acetylphosphate under vacuum supports the results obtained with the native phosphoenzyme, rather than those obtained with acetylphosphate in solution. The linkage in the active site of the phosphorylated intermediate of (Na+,K+)-ATPase appeared to be equivalent to the non-hydrated state of the model compound acetylphosphate. The phosphoenzyme with bound ouabain, or without a tightly bound divalent cation was insensitive to methylhydroxylamine. The native phosphoenzyme of (Ca2+)-ATPase was not susceptible to methylhydroxylamine.
journal_name
J Biochemjournal_title
Journal of biochemistryauthors
Ushimaru M,Shinohara Y,Fukushima Ydoi
10.1093/oxfordjournals.jbchem.a021805subject
Has Abstractpub_date
1997-09-01 00:00:00pages
666-74issue
3eissn
0021-924Xissn
1756-2651journal_volume
122pub_type
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