[Conformational flexibility of enzyme active sites]

Comparative studies of conformation and activity changes of a number of enzymes during denaturation have shown that inactivation generally precedes detectable global conformational changes. Kinetically, the inactivation rates of enzymes during denaturation are much faster than the rates of global conformational changes under identical conditions. It is suggested that the conformation of enzyme active sites are held together by weaker forces and consequently more flexible compared to the molecule as a whole. Conformational changes at the active sites were demonstrated directly by fluorescent and spin probes introduced at the active site of creatine kinase, glyceraldehyde-3-phosphate dehydrogenase and ribonuclease A. In addition, the susceptibility of ribonuclease A to proteolysis is markedly increased in dilute GuHCl. By separation and N-terminal sequence analysis of the peptide fragments liberated by hydrolysis with trypsin or proteinase K, the cleavage points can be identified to show that without gross unfolding of the RNase molecule as a whole, loosening up of active site conformation has indeed occurred during inactivation in dilute GuHCl. For the role of active site flexibility in enzyme catalysis, it is possible that each intermediate step of the whole cycle of catalysis requires the enzyme molecule to be in a different conformation state. Active site flexibility would therefore be essential for the full expression of enzyme activity. It has recently been demonstrated that conformational change, especially that at the active site, accompanies enzyme catalysis and the activation of a number of enzymes involves the loosening up of the active site structure.