Sequential Mechanisms away from Equilibrium

The derivation of rate equations for isotope exchange away from equilibrium may be understood in terms of Eq. (16.7) for the Ordered Bi Bi mechanism 

Nevertheless, isotopic exchange under nonequilibrium conditions were employed to establish the order of product release or the order of substrate addition in sequential mechanisms. 

Label from P can appear in the corresponding substrate even in the absence of Q. All that is needed is the presence of a sufficient level of EQ in the steady state. The exchange of label from Q into a substrate will not occur unless a significant concentration of P is present. Thus, one can test both products separately at levels at or above their inhibition constants, and establish the order of product release. Thus, the order of product release can be established the first product released is that which exchanges with a substrate in the absence of the another product, and the second product released is that which does not. If both products show exchange, than their release must be random. 

If the reaction is freely reversible, one can test in the same way the order of addition of substrates on the substrate side of reaction. One of the first systems to be characterized in this way was glucose-6-phosphatase (Hass Byrne, 1960). 

A more complex case was described % Nandi (1978). -Aminolevulinic acid synthase from Rhodopseudomonas spheroides is a pyridoxal phosphate-dependent enzyme that catalyzes the formation of -aminolevulinic acid from glycine and succinyl coenzyme A in this case, the release of products is random and more than one product can exchange back into substrate. 

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