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-kinase/fructose-2,6-bisphosphatase was studied by 1H-13C HMQC NMR spectroscopy of the histidine C2' and H2' nuclei. The bacterially expressed protein was specifically labeled with 13C 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-to-alanine point mutants. His-304, His-344, and His-469 exhibit 13C and 1H resonances that titrated with pH, while the remaining histidine-associated resonances did not. The 13C and 1H chemical shifts indicate that at neutral pH, His-304 and His-446 are deprotonated, while His-469 is protonated. The pKa of His-344 was determined to be 7.04. The 13C chemical shifts suggest 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 of the remaining member of the catalytic triad, Glu-327, to alanine in the resting enzyme caused an upfield shift of 1.58 and 1.30 ppm in the 1H and 13C dimensions, respectively, and significant narrowing of the His-258 cross-peak. Mutation of His-446 to alanine produced perturbations of the His-258 cross-peak that were similar to those detected in the E327A mutant. The His-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 the catalytic site of the bisphosphatase and includes specific contacts with the C-terminal regulatory region of the enzyme. The specifically 13C-labeled bisphosphatase was monitored during turnover by HMQC spectra acquired from the transient N3' phosphohistidine intermediate complex in the wild-type 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 13C and 1H resonances of this residue were shifted downfield by 1.7 and 0.31 ppm, respectively, 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 phosphohistidine was formed, generating spectra very similar to that of the wild-type enzyme in the intermediate complex. In contrast, the binding of fructose-6-phosphate, the reaction product, to the resting enzyme did not promote significant changes in the histidine-associated resonances in either the wild-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 the role of Glu-327 in the catalytic mechanism of the bisphosphatase and to identify His-446 as a putative link in the chain of molecular events that results in activation of the bisphosphatase site by cAMP-dependent phosphorylation of the hepatic bifunctional enzyme.
Okar
D A
DA
Department of Biochemistry, University of Minnesota, School of Medicine, Minneapolis 55455, USA.
Live
D H
DH
Kirby
T L
TL
Karschnia
E J
EJ
von Weymarn
L B
LB
Armitage
I M
IM
Lange
A J
AJ
D000409:Alanine / Q000737:chemistry / Q000235:genetics; D000818:Animals; D004252:DNA Mutational Analysis; D006597:Fructose-Bisphosphatase / Q000737:chemistry* / Q000235:genetics*; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000235:genetics*; D025481:Phosphofructokinase-2; D051381:Rats; D013737:Testis / Q000201:enzymology; D017186:Titrimetry
D009097:Multienzyme Complexes; D018698:Glutamic Acid; D006639:Histidine; D025481:Phosphofructokinase-2; D010732:Phosphofructokinase-1; D006597:Fructose-Bisphosphatase; D000409:Alanine; C005436:phosphohistidine