Mechanism of the bisphosphatase reaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by H-1-N-15 NMR spectroscopy

The histidines in the bisphosphatase domain of rat liver 6-phosphofructo-2- kinase/fructose-2,6-bisphosphatase were labeled with N-15, both specifical ly at N1' and globally, for use in heteronuclear single quantum correlation (HSQC) NMR spectroscopic analyses. The histidine-associated N-15 resonance s were assigned by correlation to the C2' protons which had been assigned p reviously [Okar et al., Biochemistry 38, 1999, 4471-79]. Acquisition of the H-1-N-15 HSQC from a phosphate-free sample demonstrated that the existence of His-258 in the rare N1' tautomeric state is dependent upon occupation o f the phosphate binding site filled by the O2 phosphate of the substrate, f ructose-2,6-bisphosphate, and subsequently, the phosphohistidine intermedia te. The phosphohistidine intermediate is characterized by two hydrogen bond s involving the catalytic histidines, His-258 and His-392, which are direct ly observed at the N1' positions of the imidazole rings. The N1' of phospho -His-258 is protonated (H-1 chemical shift, 14.0 ppm) and hydrogen bonded t o the backbone carbonyl of Gly-259. The N1' of cationic His-392 is hydrogen bonded (H-1 chemical shift, 13.5 ppm) to the phosphoryl moiety of the phos phohistidine. The existence of a protonated phospho-His-258 intermediate an d the observation of a fairly strong hydrogen bond to the same phosphohisti dine implies that hydrolysis of the covalent intermediate proceeds without any requirement for an "activated" water. Using the labeled histidines as p robes of the catalytic site mutation of Glu-327 to alanine revealed that, i n addition to its function as the proton donor to fructose-6-phosphate duri ng formation of the transient phosphohistidine intermediate at the N3' of H is-258, this residue has a significant role in maintaining the structural i ntegrity of the catalytic site. The H-1-N-15 HSQC data also provide clear e vidence that despite being a surface residue, His-446 has a very acidic pK( a), much less than 6.0. On the basis of these observations a revised mechan ism for fructose-2,6-bisphosphatase that is consistent with all of the prev iously published kinetic data and X-ray crystal structures is proposed. The revised mechanism accounts for the structural and kinetic consequences pro duced by mutation of the catalytic histidines and Glu-327. It also provides the basis for a hypothetical mechanism of bisphosphatase activation by cAM P-dependent phosphorylation of Ser-32, which is located in the N-terminal k inase domain.