Order of Substrate Binding

In the reaction of an enzyme with two substrates, the binding of the substrates can occur sequentially in a specific order. Thus, the binding mechanism can be divided into catalysis which proceeds through a ternary adsorption complex (enzyme -h two substrates) or through a binary complex (enzyme -h one substrate), i. e. when the enzyme binds only one of the two available substrates at a time. 

A ternary enzyme-substrate complex can be formed in two ways. The substrates are bound to the enzyme in a random fashion ( random mechanism ) or they are bound in a well-defined order ( ordered mechanism ). 

If the enzyme reacts by a random mechanism , substrates A and B form the ternary enzyme-substrate complex, EAB, in a random fashion and the P and Q products dissociate randomly from the ternary enzyme-product complex, EPQ  

Creatine kinase from muscle (cf. 12.3.6) is an example of an enzyme which reacts by a random 

In an ordered mechanism the binding during the catalyzed reaction according to equation 2.49 is as follows  

The dependence of on the fixed substrate s concentration can be used as an indicator of substrate-binding order. A fixed substrate s concentration dependence of is associated with the second substrate to bind to the enzyme. A fixed substrate s concentration independence of is associated with the first substrate to bind to the enzyme. 

---

Fromm first demonstrated how competitive inhibitors can be employed to distinguish the order of substrate binding for multisubstrate enzyme mechanisms. Each competitive inhibitor, with respect to one substrate, displays distinctive pattern(s) relative to the other substrate (s) . ... 

Initial rate enzyme kinetics are useful in defining the order of substrate binding interactions in multisubstrate... 

In a series of papers, we have proposed the torsional mechanism of energy transduction and ATP synthesis, the only unified and detailed molecular mechanism of ATP synthesis to date [16-20,56] which addresses the issues of ion translocation in Fq [16, 20, 56], ionmotive torque generation in Fq [16, 20, 56], torque transmission from Fq to Fj [17,18], energy storage in the enzyme [17], conformational changes in Fj [18], and the catalytic cycle of ATP synthesis [18, 19]. We have also studied the thermodynamic and kinetic aspects of ATP synthesis [19,20,41,42,56]. A kinetic scheme has been developed and mathematically analyzed to obtain a kinetic model relating the rate of ATP synthesis to pHjn and pH m in the Fq portion and the adenine nucleotide concentrations in the Fj portion of ATP synthase. Analysis of these kinetic models reveals a wealth of mechanistic details such as the absence of cooperativity in the Fj portion of ATP synthase, order of substrate binding and product release events, and kinetic inequivalence of ApH and Aip. 

In our model of the acetyl-CoA synthase catalytic mechanism (Figure 3), CO binds first to Cluster A (actually, the order of substrate binding is uncertain) to form a paramagnetic adduct. Fourier transform infrared... 

Use Steady-State kinetic methods to determine cat and A m for the reaction, in each direction if it is reversible, and to determine the sequence of addition of substrates and release of products. In some cases the orders of substrate binding or product release may not be easy to determine unambiguously by steady-state methods, and so further tests by transient kinetic methods may be necessary. 

The acceptance of a compulsory order of substrate binding implies that pyruvate and lactate, or their analogs, cannot bind to the enzyme in the absence of nucleotides. Tests of this prediction have been made at neutral pH. 

The most common application of exchange experiments is the determination of the order of substrate binding. Let us examine die usual Bi Bi mechanism, which can proceed via an ordered or a random pathway. 

Analysis of the reaction mechanism, using CoA substrate analogues indicates that the reaction mechanism proceeds sequentially through a ternary complex, probably by a random order of substrate binding. This proposal requires verification using the in vivo, acyl-ACP, substrates. 

---