For the U-system model, all rate constants and kinetic orders of substrates and enzymes were set to 1, while the kinetic orders of parameters for inhibitions and activations were set to be -0.5 and 0.5, respectively.
The enzyme has a fixed order of substrate binding, with ATP binding first, followed by HMDP.
The enzyme deglycosylation induces significant changes in the order of substrate selectivity for gAmPDH and dgAmPDH.
Initial-rate kinetic experiments are then employed to reveal optimal reaction conditions, to evaluate the Michaelis constant, Km, and maximal velocity, Vm, to establish the order of substrate addition and product release, and to define the basic modes of inhibitor and activator action.
From these crystal structures, a sequential order of substrate binding was postulated.
To explore the mechanism of formation of the ternary complex, i.e. the order of substrate binding, two sets of experiments were designed.
Here, the order of substrate binding as well as the underlying conformational changes were investigated by NMR confirming the model derived from the crystal structures.
Thus, the order of substrate binding to OcDH as well as the molecular signals involved in octopine formation can now be described in molecular detail.
Since conformational dynamics in the ES1 state is very slow, this order of substrate binding (S1 first, then S2) would result in slower catalytic rate as compared to a different order (S2 first, then S1).
Data from enzyme kinetics experiments favoring an ordered mechanism as well as a random mechanism have been reported warranting further studies on the order of substrate binding to CaMKII [13] [15], [23].
