Structural and Regulatory Studies on Rabbit Muscle Phosphofructokinase.

Rabbit muscle phosphofructokinase (EC 2.7.1.11; PFK) undergoes a time dependent lag in activity before attaining maximal catalytic velocity. This phenomenon, termed hysteresis, is evident when the enzymatic reaction is initiated with one of the substrates, fructose 6-phosphate (F 6-P). The lag is not manifested with initiation of the reaction by the other substrate, ATP, or with the enzyme. The length of the lag was found to be dependent upon enzyme concentration, pH, concentration of F 6-P, and the presence of inhibitors or activators in the incubation medium. A model is proposed which ascribes this phenomenon to a bisphosphate induced conformational change of the enzyme to a more active state. Results with locked, isosteric analogues of fructose 1,6-bisphosphate (F 1,6-P(,2)) suggest that there are two distinct phases in the hysteretic process in PFK--reduction of the lag and actual enhancement of catalytic velocity. The former appears to show no anomeric preference, while the latter appears to be modulated by the (alpha)-anomer of F 1,6-P(,2). A series of straight chain bisphosphate compounds were synthesized and studied as models for fructose 2,6-bisphosphate (F 2,6-P(,2)), a potent activator of PFK. 1,4-Butanediol bisphosphate, which approximates the spacing across the PO-C(,6)-C(,5)-O-C(,2)-OP portion of the F 2,6-P(,2) molecule, was found to be quite effective in activating PFK. The activation constant for 1,4-butanediol bisphosphate was determined to be 6.6 x 10(\u27-6) M, compared to 1.2 x 10(\u27-7) M for F 2,6-P(,2). The data suggest that the allosteric bisphosphate binding site of PFK requires the distance between the two phosphate groups of the activator be in the range of 9.0-10.2 (ANGSTROM). 1,4-Butanediol bisphosphate was also found to protect the enzyme against inhibition by citrate. Treatment of PFK with the arginine specific reagent, phenylglyoxal, led to inactivation of the enzyme. Amino acid analysis determined that a least four arginyl residues were modified. Complete loss of activity was correlated with modification of six arginyl residues. F 6-P and F 1,6-P(,2) protected against inactivation, with two less arginyl residues being modified. While ATP, ADP, and AMP also protected against inactivation; neither was able to decrease the number of arginyl residues modified. These data indicate that arginyl residues are essential for binding at the active site of PFK