Abstract：
:The dynamic equilibrium of a catalytic site between active and inactive conformations, the missing link between the structure and function of allosteric enzymes, was identified using protein engineering and NMR techniques. Kinetic analyses of the wild-type and three mutants of Thermus L-lactate dehydrogenase established that the allosteric property of the enzyme is associated with a concerted transition between the high-affinity (R) and low-affinity (T) states. By introducing mutations, we prepared an enzyme in which the R and T states were balanced. The conformation of the enzyme-bound coenzyme, NAD+, which interacts directly with the substrate, was analyzed using NMR spectroscopy. NAD+ bound to the mutant enzyme was in a conformational mixture of the active and inactive forms, while NAD+ took on predominantly one of the two forms when it was bound to the other enzymes we had analyzed. We interpret this to mean that the catalytic site is in equilibrium between the two conformations. The ratio of the conformers of each enzyme agreed with the [T]/[R] ratio as determined by kinetic analyses. Therefore, it is the identified conformational equilibrium of the catalytic site that governs the allosteric regulation of the enzyme activity.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Koide S,Yokoyama S,Matsuzawa H,Miyazawa T,Ohta Tdoi
10.1021/bi00138a017subject
Has Abstractpub_date
1992-06-16 00:00:00pages
5362-8issue
23eissn
0006-2960issn
1520-4995journal_volume
31pub_type
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