The Fructose 6-Phosphate Site of Phosphofructokinase
Year: 1974
Type: article
Abstract: The phosphoryl acceptor substrate of phosphofructokinase (EC 2.7.1.11), namely d-fructose-6-P, can exist in four forms in solution due to facile ring-chain tautomerization, anomerization, and hydration. These forms include the cyclic α- and β-furanose anomers and the acyclic keto and hydrated keto forms. In order to establish the tautomeric and anomeric specificity of phosphofructokinase (from rabbit muscle), several structurally locked, isosteric analogues of the four forms of d-fructose-6-P were tested kinetically for enzymatic activity. 2,5-Anhydro-d-mannitol-1-P (structurally identical with 2,5-anhydro-d-mannitol-6-P) and 2,5-anhydro-d-glucitol-6-P were synthesized and characterized. The former, a novel analogue of β-d-fructofuranose-6-P, was found to be an alternate substrate with kinetic constants closer to the natural substrate than any compound yet reported (Km = 0.41 mm and a Vmax relative to fructose-6-P = 87%). The latter, an analogue of α-d-fructofuranose-6-P, was found to be an effective competitive inhibitor of phosphofructokinase (Ki = 0.34 mm). d-Xylulose-5-P, d-mannitol-1-P (identical with d-mannitol-6-P), d-glucitol-6-P, and dihydroxyacetone, good analogues of the acyclic keto form of d-fructose-6-P and its hydrate, were inactive as inhibitors or alternate substrates. These findings indicate that, in binding, the fructose-6-P site has a tautomeric specificity for the cyclic furanose forms of its substrate, whereas in phosphorylation, it has a more restrictive anomeric specificity, accepting only β-d-fructofuranose-6-P. It seems that phosphofructokinase has evolved so as to phosphorylate the form of its substrate most abundant in solution. It is proposed that this enzyme be more accurately renamed ATP:β-d-fructofuranose 6-phosphate 1-phosphotransferase.
Source: Journal of Biological Chemistry
Institution: Louisiana State University
Language: English
Subfield: Organic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W1510102401