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Hydrogen bonding in the fructose‐2,6‐bisphosphatase reaction correlates with activity
Author(s) -
Okar David Anthony,
Zangger Klaus
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1014-b
Subject(s) - tautomer , chemistry , hydrogen bond , protonation , nuclear magnetic resonance spectroscopy , fructose 1,6 bisphosphatase , stereochemistry , catalysis , active site , fructose , biochemistry , molecule , organic chemistry , ion
The bifunctional enzyme, 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase, and the associated biofactor, fructose‐2,6‐bisphosphate (F‐2,6‐P 2 ) are important regulators of carbohydrate metabolism. We have used multi‐dimensional NMR spectroscopy to determine the tautomeric and protonation states of the catalytic histidines in the bisphosphatase domain from human heart (hhFBP‐2). Resonances were assigned by comparison to those conserved in the rat liver bisphosphatase (rlFBP‐2). The FBP‐2 reaction utilizes a stable phosphohistidine intermediate that can be observed by NMR spectroscopy. In resting hhFBP‐2 the phospho‐acceptor, His‐257, exists as the neutral N1′ tautomer, the other catalytic residue, His‐391, is cationic, as is His‐419, while His‐445 is in the neutral N3′ tautomeric form. The 13 C and 1 H chemical shifts assigned to the catalytic histidines in hhFBP‐2 are consistently up‐field of the corresponding resonances in rlFBP‐2, particularly in the transient reaction intermediate. These reveal isozyme‐specific changes in the bisphosphatase active site that suggest weaker hydrogen bonds within the cardiac bisphosphatase transient intermediate. Because these hydrogen bonds stabilize the phosphohistidine intermediate, the observed differences can be related to physiologically appropriate differences in the FBP‐2 activity in liver and heart.