Rat liver 6‐phosphofructo 2‐kinase/ fructose 2,6‐bisphosphatase: A review of relationships between the two activities of the enzyme

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Abstract
Both the synthesis and the degradation of Fru‐2,6‐P 2 are catalyzed by a single enzyme protein; ie, the enzyme is bifunctional. This protein, which we have designated 6‐phosphofructo 2‐kinasc/fructose 2,6‐bisphosphatase is an important enzyme in the regulation of hepatic carbohydrate metabolism since its activity determines the steady‐slate concentration of fructose 2,6‐P2, an activator of 6‐phosphofructo 1‐kinase and an inhibitor of fructose 1,6‐bisphosphatase. Regulation of the bifunctional enzyme in intact cells is a complex function of both covalent modification via phosphorylation/dephosphorylation and the influence of substrates and low molecular weight effectors. Recent evidence suggests that both reactions may proceed by two‐step transfer mechanisms with different phosphoenzyme intermediates. The enzyme catalyzes exchange reactions between ADP and ATP and between fructose 6‐P and fructose 2,6‐P2. A labeled phosphoenzyme is formed rapidly during incubation with [2‐32P]Fru‐2,6‐P2. The labeled residue has been identified as 3‐phosphohistidinc. However, it was not possible to demonstrate significant labeling of the enzyme directly from [γ‐32P]ATP. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E‐P is labile) and a fructose 2,6‐bisphosphatase site which is readily phosphorylated by fructose 2,6‐P2. Additional evidence in support of two active sites include: (1) limited proteolysis with thermolysin results in loss of 6‐phosphofructo 2‐kinase activity and activation of fructose 2,6‐bisphosphatase, (2) mixed function oxidation results in inactivation of the 6‐phosphofructo 2‐kinase but no affect on the fructose 2,6‐bisphosphatase, (3) N‐ethylmaleimide treatment also inactivates the kinase but docs not affect the bisphosphatase, and (4) p‐chlorom‐ercuribenzoate immediately inactivates the fructose 2,6‐bisphosphatase but not the 6‐phosphofructo 2‐kinasc. Our findings indicate that the bifunctional enzyme is a rather complicated enzyme; a dimer, probably with two catalytic sites reacting with sugar phosphate, and with an unknown number of regulatory sites for most of its substrates and products. Three enzymes from Escherichia coli, isocitric dehydrogenase kinase/phosphatase, glutamine‐synthetase adenylyltransferase, and the uridylyltransferase for the regulatory protein PU in the glutamine synthetase cascade system also catalyze opposing reactions probably at two discrete sites. All four enzymes are important in the regulation of metabolism and may represent a distinct class of regulatory enzymes.

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