Conformation change product dissociation, 181-2 substrate

Fig. 2. A pictorial representation of the ribonuclease reaction. The free enzyme (A) exists in two conformational states differing by small movements of the hinge region joining the two halves of the molecule. The substrate is bound (B) and a conformational change occurs closing the hinge (C). Concerted acid-base catalysis then occurs (D) products are formed ( ) the conformational change is reversed (F) and product(s) dissociate to give free enzyme.

Fig. 8.4. Reaction in the enzyme active catalytic site. A. The enzyme contains an active catalytic site, shown in dark blue, with a region or domain where the substrate binds. The active site also may contain cofactors, nonprotein components that assist in catalysis. B. The substrate forms bonds with amino acid residues in the substrate binding site, shown in light blue. Substrate binding induces a conformational change in the active site. C. Functional groups of amino acid residues and cofactors in the active site participate in forming the transition state complex, which is stabilized by additional noncovalent bonds with the enzyme, shown in blue. D. As the products of the reaction dissociate, the enzyme returns to its original conformation.

This enzyme requires ordered addition of its substrates - the nucleotide has to bind prior to lactate or pyruvate. Similarly the nucleotide dissociates after the other substrate. The conformation change to form the reactive complex occurs when both NADH and pyruvate are bound. In the reverse direction, when the concentration of free pyruvate is negligible, an isomerization step (E E) has to occur after pyruvate has dissociated, but before NADH can dissociate. Therefore an additional step is involved in NADH dissociation after catalytic turnover. Some evidence for two step binding of NADH to lactate dehydrogenase has been found by Wu etal. 99 ) even in the absence of pyruvate. Similar phenomena are observed during the dissociation of the products after ATP hydrolysis by myosin (see section 5.1). Some of these events may still be subject to revision, but it is clear that product dissociation from enzymes requires quite detailed analysis. Some of the approaches to this problem have been outlined in section 5.2. 

It is important to stress that I have used the term tight binding of the E-S complex in this context to refer only to the rate at which the substrate dissociates from the enzyme relative to the rate of reaction to form products. It is often the case that the binding of substrate in an initial, weak collision complex induces a change in enzyme conformation, resulting in a slower rate of substrate release and tighter binding 29. 40). Nonetheless, it is important to keep in mind the... 

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