guanosine-diphosphate and fructose-6-phosphate

The kinetic mechanism of phosphofructokinase from Bacillus sterothermophilus has been investigated using steady-state measurements. The double-reciprocal patterns observed for initial velocity, product inhibition, and mixed alternate substrate studies of the reverse reaction establish 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 patterns for the forward reaction show significant nonlinearity and resemble those seen for competitive substrate (MgATP) inhibition of an enzyme 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 effector site. Product and dead-end inhibition studies of the forward reaction are consistent with a random mechanism, after taking into account the effects of substrate inhibition by MgATP. Initial velocity measurements 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 proceeds through two alternative pathways, one of which is kinetically disfavored. The observed MgATP substrate inhibition arises from both reaction flux through the disfavored pathway and, to some extent, abortive binding of MgATP in the Fru-6P site.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Binding Sites; Binding, Competitive; Fructosediphosphates; Fructosephosphates; Geobacillus stearothermophilus; Guanosine Diphosphate; Kinetics; Mutagenesis, Site-Directed; Phosphofructokinase-1; Structure-Activity Relationship

Tetrameric Escherichia coli phosphofructokinase dissociates reversibly on incubation under hydrostatic pressures of 80 MPa and above, yielding inactive dimers and monomers. The transition is dependent upon enzyme concentration and presence of ligands. The substrate, D-fructose 6-phosphate, which bridges the intersubunit interface at the active site, produces a massive stabilization to pressure, whereas ATP, which binds to only one subunit, induces only a mild stabilization. Both the positive allosteric regulator, GDP, and the negative allosteric regulator, phosphoenolpyruvate, whose binding sites lie at the other subunit interface, produce an intermediate effect. Of these ligands, only ATP increases the rate of reactivation after depressurization.

Topics: Adenosine Triphosphate; Binding Sites; Enzyme Activation; Enzyme Reactivators; Escherichia coli; Fructosephosphates; Guanosine Diphosphate; Hydrostatic Pressure; Kinetics; Macromolecular Substances; Phosphoenolpyruvate; Phosphofructokinase-1

In the presence of its allosteric activator GDP, the major phosphofructokinase-1 from Escherichia coli K12 follows Michaelis-Menten kinetics. The kinetic behavior observed at steady-state using different concentrations of the substrates ATP and fructose-6-phosphate and the pattern of inhibition by the substrate analogs adenylyl-(beta, gamma-methylene)-diphosphonate and D-arabinose-5-phosphate are consistent with a random sequential mechanism in rapid equilibrium, rather than with an ordered binding as was suggested earlier. However, ATP and fructose-6-phosphate do not bind independently to the same active site, since the apparent affinity for one substrate is decreased about 20-fold when the other substrate is already bound. The antagonism between ATP and fructose-6-phosphate shows that a negative interaction occurs during the reaction with E. coli phosphofructokinase-1 which must be considered in addition to its allosteric properties.

Topics: Adenosine Triphosphate; Allosteric Regulation; Escherichia coli; Fructosephosphates; Guanosine Diphosphate; Kinetics; Pentosephosphates; Phosphofructokinase-1

Crystal structures of the high- and low-activity states of the allosteric enzyme phosphofructokinase implicate three arginines in substrate binding, catalysis and cooperativity. Arginines 162 and 243 reach into the active site from an adjacent subunit and interact with the cooperative substrate fructose 6-phosphate. Mutation of these arginines to serine results in mutant enzymes with reduced substrate binding and lowered cooperativity, but with little change in their catalytic ability (kcat). Arg 72 bridges the two substrates fructose 6-phosphate and ATP, and interacts with the 1-phosphate of the product fructose 1,6-biphosphate. Mutation of this residue to serine reduces the catalytic activity, cooperativity and binding of fructose 6-phosphate and fructose 1,6-bisphosphate. In the reverse reaction, the kinetics of wild-type and the Ser 72 mutant with respect to fructose 1,6-bisphosphate are hyperbolic, whereas those of the Ser 162 and Ser 243 mutants are sigmoidal. These results show that each of the three arginines contributes to cooperativity and to the transmission of allosteric signals between the four subunit of the enzyme.

Topics: Adenosine Triphosphate; Allosteric Regulation; Arginine; Binding Sites; Escherichia coli; Fructosephosphates; Guanosine Diphosphate; Kinetics; Molecular Structure; Mutation; Phosphofructokinase-1; Protein Conformation; Structure-Activity Relationship

Limited proteolysis of Escherichia coli phosphofructokinase by subtilisin yields a homogeneous derivative. This proteolyzed protein is composed of four polypeptide chains, with a molecular weight of 32 000 as compared to 37 000 for the original enzyme. Removal on each chain of about 5 kdaltons maintains the enzymatic activity and does not change the apparent affinity for the substrates ATP and fructose 6-phosphate. Limited proteolysis, however, affects the cooperativity of fructose 6-phosphate binding: the Hill coefficient is reduced from almost 4 in the native enzyme to only 2 in its proteolyzed derivative. Also, the proteolyzed protein is no longer sensitive to allosteric effectors, activator, or inhibitor. These changes in regulatory properties upon proteolysis are apparently due to the destruction of the effector binding site. The allosteric effector GDP protects phospho-fructokinase against proteolysis and irreversible thermal inactivation; GDP is, however, inefficient in protecting the proteolyzed protein against thermal denaturation. These results suggest that phosphofructokinase may function as a dimer of dimers, in which homotropic and heterotropic allosteric effects are not mediated by the same sets of quaternary interactions.

Topics: Adenosine Triphosphate; Escherichia coli; Fructosephosphates; Guanosine Diphosphate; Hot Temperature; Macromolecular Substances; Molecular Weight; Phosphofructokinase-1; Protein Denaturation; Subtilisins