KINETIC CHARACTERISTICS OF PHOSPHOFRUCTOKINASE FROM BACILLUS-STEAROTHERMOPHILUS - MGATP NONALLOSTERICALLY INHIBITS THE ENZYME

The kinetic mechanism of phosphofructokinase from Bacillus sterothermo philus has been investigated using steady-state measurements. The doub le-reciprocal patterns observed for initial velocity, product inhibiti on, and mixed alternate substrate studies of the reverse reaction esta blish that the mechanism involves rapid-equilibrium random binding of substrates and the formation of an abortive complex composed of enzyme , MgADP, and fructose 6-phosphate (E-MgADP-Fru-6P). Initial velocity p atterns for the forward reaction show significant nonlinearity and res emble those seen for competitive substrate (MgATP) inhibition of an en zyme that obeys a random mechanism. A mutant BsPFK enzyme (GV212) was used to show that the inhibition is not due to MgATP binding in the ef fector site. Product and dead-end inhibition studies of the forward re action are consistent with a random mechanism, after taking into accou nt the effects of substrate inhibition by MgATP. Initial velocity meas urements at low MgATP concentration show that the binding of MgATP is not a rapid-equilibrium process; i.e., the rate of catalysis is faster than the rate of substrate binding. It is concluded that the kinetic mechanism of the forward reaction is sequential random, with the rate of MgATP binding slower than the catalytic rate. A model is presented that incorporates these results and proposes that substrate binding pr oceeds through two alternative pathways, one of which is kinetically d isfavored. The observed MgATP substrate inhibition arises from both re action flux through the disfavored pathway and, to some extent, aborti ve binding of MgATP in the Fru-6P site.