Evidence for a specific phosphoryl binding site in swine kidney phosphofructokinase.

Abstract

Phosphofructokinase (PFK) from swine kidney was purified by a procedure which included affinity chromatography on Cibacron blue F3GA-Sepharose 4B and ATP-Sepharose 4B columns in order to examine its binding properties. The homogeneous enzyme was purified more than 3000-fold with a yield of 30% and it had a specific activity of 39.8 mumol/min/mg of protein at 25 degrees C. The molecular weight of the native enzyme was 360 000 and it contained 4 identical subunits of molecular weight 88 000. The principal catalytically reacting form of the enzyme had a S20,w of 13.7 S which corresponds to a molecular weight of 360 000 +/- 6 000. The initial velocity patterns in the forward and reverse directions suggested a sequential mechanism for the reaction. The Km values for fructose 6-phosphate, ATP, fructose, 1,6-bisP and ADP were 33 microM, 8.3 microM, 460 microM and 110 microM, respectively. The homogeneous native enzyme binds specifically to phosphoryl groups immobilized in cellulose phosphate columns. ATP and fructose 6-phosphate interacted with the enzyme and decreased its affinity for phosphoryl binding sites. Other metabolites including fructose 1,6-bisP, glucose 6-phosphate and various nucleotides, alone or in various combinations, were ineffective in promoting the dissociation of the enzyme. Allosteric effectors of the enzyme, such as citrate and AMP were also inactive. However, the cooperatively altered the concentration of ATP required to dissociate the enzyme from phosphoryl groups. The bound enzyme was enzymatically inactive. The enzyme was also inactivated when it was treated with pyridoxal 5'-phosphate and reduced with sodium borohydride and the inactive enzyme no longer bound to cellulose phosphate. These effects were not observed when treatment with pyridoxal 5'-phosphate was carried out in the presence of fructose 6-phosphate. These observations and the results of similar studies with swine kidney fructose 1,6-bisphosphatase (FBPase) show that both enzymes share the unique property of binding specifically to phosphoryl groups. FBPase interacts through its allosteric AMP binding site and PFK binds through its fructose 6-P binding site. This specific binding of both enzymes through these sites result in the inactivation of PFK and the desensitization of FBPase to allosteric inhibition by AMP. In the unbound state PFK may be active and FBPase can be inhibited by AMP. Taken collectively, these binding effects could play a role in the reciprocal regulation of these enzymes during gluconeogenesis in kidney.