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REVIEW: The Role of Active Site Flexibility in Enzyme Catalysis

Chen-Lu Tsou

National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, China; fax: +86-10-6202-2026; E-mail: cltsou@sun5.ibp.ac.cn

Received July 24, 1997
It has been shown in this and other laboratories that during the unfolding of a number of enzymes inactivation generally precedes global unfolding of the enzyme molecule, leading to the suggestion that enzyme active sites are usually more "fragile" and more easily "perturbed" than the molecule as a whole and are therefore conformationally more flexible than the rest of the molecule. However, the role of active site flexibility in enzyme catalysis still remains to be explored. In the induced fit hypothesis originally proposed by Koshland, the presence of the substrate induces a conformational change at the active site so as to fit with the structure of the substrate. By X-ray crystallographic structural analysis of E. coli dihydrofolate reductase liganded with cofactors and substrates, Sawaya and Kraut showed the enzyme in different conformational states indeed while complexed with different ligands, suggesting that the enzyme molecule passes through different conformational states through the whole process of catalysis. Muscle lactate dehydrogenase can be stabilized either in concentrated ammonium sulfate or by cross-linking with glutaraldehyde together with a decrease in enzyme activity which can be restored to the original level in dilute guanidine hydrochloride possibly by increased flexibility at the active site. It is known that a number of enzymes can be activated by chaotropic agents such as urea or guanidine hydrochloride. The activation of dihydrofolate reductase by either urea or guanidine hydrochloride is accompanied by an increase in susceptibility to proteolysis. Isolation of the tryptic peptides of the activated enzyme and sequence analysis allowed identification of the sites of proteolysis to be at or near the active site of the enzyme, indicating an opening up of the active site conformation in the activated state. All the above indicate that active site flexibility plays an important role in enzyme catalysis. It is possible that during the catalytic cycle, the enzyme molecule passes through different stages and each stage requires the molecule to be in a different conformation, especially at the active site. Rapid transition between the different conformational states, and hence the flexibility of the active site, is therefore mandatory for the maximal expression of enzyme activity.
KEY WORDS: enzyme catalysis, flexibility of active site, enzyme inactivation, dihydrofolate reductase, lactate dehydrogenase