Binding-site mechanism

It is instructive to write this equation in the form analogous to that for a single agonist binding site mechanism,... 

The relationship between popm (pconlrol) and agonist concentration for the two-agonist binding site mechanism is given in Eq. (6.4) and reproduced below in a slightly different form ... 

The dependence of chiral recognition on the formation of the diastereomeric complex imposes constraints on the proximity of the metal binding sites, usually either an hydroxy or an amine a to a carboxyHc acid, in the analyte. Principal advantages of this technique include the abiHty to assign configuration in the absence of standards, enantioresolve non aromatic analytes, use aqueous mobile phases, acquire a stationary phase with the opposite enantioselectivity, and predict the likelihood of successful chiral resolution for a given analyte based on a weU-understood chiral recognition mechanism. 

The basic kinetic properties of this allosteric enzyme are clearly explained by combining Monod s theory and these structural results. The tetrameric enzyme exists in equilibrium between a catalytically active R state and an inactive T state. There is a difference in the tertiary structure of the subunits in these two states, which is closely linked to a difference in the quaternary structure of the molecule. The substrate F6P binds preferentially to the R state, thereby shifting the equilibrium to that state. Since the mechanism is concerted, binding of one F6P to the first subunit provides an additional three subunits in the R state, hence the cooperativity of F6P binding and catalysis. ATP binds to both states, so there is no shift in the equilibrium and hence there is no cooperativity of ATP binding. The inhibitor PEP preferentially binds to the effector binding site of molecules in the T state and as a result the equilibrium is shifted to the inactive state. By contrast the activator ADP preferentially binds to the effector site of molecules in the R state and as a result shifts the equilibrium to the R state with its four available, catalytically competent, active sites per molecule. 

The overall direction of the reaction will be determined by the relative concentrations of ATP, ADP, Cr, and CrP and the equilibrium constant for the reaction. The enzyme can be considered to have two sites for substrate (or product) binding an adenine nucleotide site, where ATP or ADP binds, and a creatine site, where Cr or CrP is bound. In such a mechanism, ATP and ADP compete for binding at their unique site, while Cr and CrP compete at the specific Cr-, CrP-binding site. Note that no modified enzyme form (E ), such as an E-PO4 intermediate, appears here. The reaction is characterized by rapid and reversible binary ES complex formation, followed by addition of the remaining substrate, and the rate-determining reaction taking place within the ternary complex. 

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