Rabbit Muscle Phosphofructokinase: I. ANOMERIC SPECIFICITY; INITIAL VELOCITY KINETICS

Abstract

Abstract Initial velocity patterns have been obtained for rabbit muscle phosphofructokinase at pH 7.4, 25°, with six nucleotides as substrates, and also with 2,5-anhydro-d-mannitol-6-P and 2,5-anhydro-d-mannitol-1,6-P2 as substrate analogs. In the forward (MgNTP, fructose-6-P) direction most of the patterns were parallel or nearly so, but with MgITP and MgCTP there was clear convergence. In the reverse direction all patterns except for MgGDP were clearly intersecting. The mechanism must therefore be sequential. The good activity of the anhydromannitol analog, but the failure of l-sorbose-6-P or 2,5-anhydro-d-glucitol-6-P prepared either chemically or enzymatically to act as a substrate suggests that the enzyme is specific both for the β anomers of fructose-6-P and fructose-P2 and for the d configuration at carbon 5. The activity of fructose-1-P (presumably the β anomer) as a substrate is consistent with this view, as is the failure of l-sorbose-1-P to act either as inhibitor or substrate.