Substrate binding conformation change prior

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. 

Prior to the elucidation of these structures, it was known that NOS must dimerize through the oxygenase domain for catalytic function [99]. Structures of the oxygenase dimer of both iNOS and eNOS reveal that dimer formation reinforces the substrate binding channel and sequesters two pterin molecules within two symmetry-related lariats (see Figure 11) [100-102]. The dimer interface is extensive with between 1200 and 2800 of buried surface [100-102]. Additionally, conformational changes upon dimerization of iNOS expose the heme edge opposite of the eenter channel, and provide a possible interaction surface for the complementarily shaped reduetase domain [100]. 

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