The roles of Glu-327 and His-446 in the bisphosphatase reaction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by NMR spectroscopic and mutational analyses of the enzyme in the transient phosphohistidine intermediate complex

The bisphosphatase domain derived from the rat liver 6-phosphofructo-2-kina se/fructose-2,6-bisphosphatase was studied by H-1-C-13 HMQC NMR spectroscop y of the histidine C2' and H2' nuclei. The bacterially expressed protein wa s specifically labeled with C-13 at the ring C2' position of the histidines . Each of the seven histidine residues gave rise to a single cross-peak in the HMQC spectra, and these were assigned by use of a series of histidine-t o-alanine point mutants. His-304, His-344, and His-469 exhibit C-13 and H-1 resonances that titrated with pH, while the remaining histidine-associated resonances did not. The C-13 and H-1 chemical shifts indicate that at neut ral pH, His-304 and His-446 are deprotonated, while His-469 is protonated. The pK(a)* of His-344 was determined to be 7.04. The C-13 chemical shifts s uggest that the deprotonated His-258 exists as the N1' tautomer, while His- 392 and His-419 are protonated in the resting, wild-type enzyme. Mutation o f the remaining member of the catalytic triad, Glu-327, to alanine in the r esting enzyme caused an upfield shift of 1.58 and 1.30 ppm in the H-1 and C -13 dimensions, respectively, and significant narrowing of the His-258 cros s-peak. Mutation of His-446 to alanine produced perturbations of the His-25 8 cross-peak that were similar to those detected in the E327A mutant. The H is-392 resonances were also shifted by the E327A and H446A mutations. These observations strongly suggest that residues His-258, Glu-327, His-392, and His-446 exist within a network of interacting residues that encompasses th e catalytic site of the bisphosphatase and includes specific contacts with the C-terminal regulatory region of the enzyme. The specifically C-13-label ed bisphosphatase was monitored during turnover by HMQC spectra acquired fr om the transient N3' phosphohistidine intermediate complex in the wildtype enzyme, the E327A mutant, and the H446A mutant. These complexes were formed during reaction with the physiological substrate fructose-2,6-bisphosphate . Upon formation of the phosphohistidine at His-258, the C-13 and H-1 reson ances of this residue were shifted downfield by 1.7 and 0.31 ppm, respectiv ely, in the wild-type enzyme. The upfield shifts of the His-258 resonances in the E327A and H446A mutant resting enzymes were reversed when the phosph ohistidine was formed, generating spectra very similar to that of the wild- type enzyme in the intermediate complex. In contrast, the binding of fructo se-6-phosphate, the reaction product, to the resting enzyme did not promote significant changes in the histidine-associated resonances in either the w ild-type or the mutant enzymes. The interpretation of these data within the context of the X-ray crystal structures of the enzyme is used to define th e role of Glu-327 in the catalytic mechanism of the bisphosphatase and to i dentify His-446 as a putative link in the chain of molecular events that re sults in activation of the bisphosphatase site by cAMP-dependent phosphoryl ation of the hepatic bifunctional enzyme.