Dynamics of local conformation and enzyme function.
Enzyme function is critically affected by gross changes in tertiary structure, but much less is known about the dependence of activity on localized conformational changes that might occur during the catalytic process. Such information is essential to the understanding and verification of enzyme mechanisms, and can only be obtained from systems in which the substrate binding and catalytic groups of the enzyme active centre are not only identified but also rendered appropriate probes of the catalytic reaction, that is they must signal changes in their mutual interactions at rates at least as fast as catalysis. It should then be possible to observe directly each of the dynamic events that result in catalysis. Time-averaged structural analyses can neither reveal the dynamics of catalysis nor describe the conformational details of molecules who structures-particularly of the active centre-are motile. Moreover, the three-dimensional structures of enzymes in crystals and solutions may not always be identical as evidenced by marked differences between the kinetic properties of crystalline and of dissolved enzymes (e.g. hexokinase, glycogen phosphorylase and carboxypeptidases A and B). Various experimental approaches have been devised to explore the relationship between structure and function of these and other enzymes. We have focused on the syncatalytic, spatial relationships of active-site residues as they bear on the mechanism of enzyme action using absorption, circular dichroism, magnetocircular dichroic, and resonance Raman spectroscopy as well as resonance energy transfer. Stopped-flow, pH and temperature jump methods quantitatively assess both the conformational and local structural features of the enzyme and reveal multiple, discrete conformational states that prove to have mechanistic significance. These and related data serve as a basis for a minimal model of the dynamic aspects of enzyme action.
D001665:Binding Sites; D002268:Carboxypeptidases; D002942:Circular Dichroism; D003035:Cobalt; D003460:Crystallization; D004798:Enzymes / Q000378:metabolism; D007700:Kinetics; D008670:Metals; D011485:Protein Binding; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity
D004798:Enzymes; D008670:Metals; D003035:Cobalt; D002268:Carboxypeptidases