pyrophosphate and 3-phosphoglycerate

ADP-glucose pyrophosphorylase catalyzes the synthesis of ADP-glucose (ADP-Glc) from Glc-1-phosphate (G-1-P) and ATP. Kinetic studies were performed to define the nature of the reaction, both in the presence and absence of allosteric effector molecules. When 3-phosphoglycerate (3-PGA), the putative physiological activator, was present at a saturating level, initial velocity studies were consistent with a Theorell-Chance BiBi mechanism and product inhibition data supported sequential binding of ATP and G-1-P, followed by ordered release of pyrophosphate and ADP-Glc. A sequential mechanism was also followed when 3-PGA was absent, but product inhibition patterns changed dramatically. In the presence of 3-PGA, ADP-Glc is a competitive inhibitor with respect to ATP. In the absence of 3-PGA--with or without 5.0 mm inorganic phosphate--ADP-Glc actually stimulated catalytic activity, acting as a feedback product activator. By contrast, the other product, pyrophosphate, is a potent inhibitor in the absence of 3-PGA. In the presence of subsaturating levels of allosteric effectors, G-1-P serves not only as a substrate but also as an activator. Finally, in the absence of 3-PGA, inorganic phosphate, a classic inhibitor or antiactivator of the enzyme, stimulates enzyme activity at low substrate by lowering the K(M) values for both substrates.

Topics: Diphosphates; Endosperm; Glucose-1-Phosphate Adenylyltransferase; Glyceric Acids; Molecular Sequence Data; Plant Proteins; Substrate Specificity; Zea mays

3-Phosphoglycerate kinase (PGK) is a two-domain hinge-bending enzyme. It is still unclear how the geometry of the active site is formed during domain closure and how the catalytic residues are brought into the optimal position for the reaction. Comparison of the three-dimensional structures in various open and closed conformations suggests a large (10 A) movement of Lys 215 during domain closure. This change would be required for direct participation of this side chain in both the catalyzed phospho transfer and the special anion-caused activation. To test the multiple roles of Lys 215, two mutants (K215A and K215R) were constructed from human PGK and characterized in enzyme kinetic and substrate binding studies. For comparison, mutants (R38A and R38K) of the known essential residue, Arg 38, were also produced. Drastic decreases (1500- and 500-fold, respectively), as in the case of R38A, were observed in the kcat values of mutants K215A and K215R, approving the essential catalytic role of Lys 215. In contrast, the R38K mutation caused an only 1.5-fold decrease in activity. This emphasizes the importance of a very precise positioning of Lys 215 in the active site, in addition to its positive charge. The side chain of Lys 215 is also responsible for the substrate and anion-dependent activation, since these properties are abolished upon mutation. Among the kinetic constants mainly the Km values of MgATP and 1,3-BPG are increased (approximately 20- and approximately 8-fold, respectively) in the case of the neutral K215A mutant, evidence of the interaction of Lys 215 with the transferring phospho group in the functioning complex. Weakening of MgATP binding (a moderate increase in Kd), but not of MgADP binding, upon mutation indicates an initial weak interaction of Lys 215 with the gamma-phosphate already in the nonfunctioning open conformation. Thus, during domain closure, Lys 215 possibly moves together with the transferring phosphate; meanwhile, this group is being positioned properly for catalysis.

Topics: Adenosine Triphosphate; Allosteric Regulation; Allosteric Site; Amino Acid Substitution; Calorimetry, Differential Scanning; Catalysis; Catalytic Domain; Circular Dichroism; Diphosphates; Diphosphoglyceric Acids; Enzyme Stability; Glyceric Acids; Humans; Kinetics; Lysine; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Phosphoglycerate Kinase; Protein Conformation; Recombinant Proteins; Substrate Specificity; Sulfhydryl Compounds