Catalytic properties, stability and the structure of the conformational lock in the alkaline phosphatase from Escherichia coli

Abstract

The activity of oligomeric enzymes is sensitive to the formation and dissociation of the interprotein contacts that make up the conformational lock. The mechanism for this is discussed in this article concerning the alkaline phosphatase (AP) from Escherichia coli. Study of the AP from various source shows that the thermoinactivation curves, obtained under various conditions, have induction periods that may be ascribed to latent structural changes in the conformational lock. The analysis of kinetic curves has allowed us to calculate the minimum number of denaturation stages in the conformational lock ( n=3), i.e., the stable dimer becomes labile and capable of dissociation by the sequential dissociation of two of the three contacts which take part in the conformational lock. Three-dimensional structural analysis of AP from E. coli established that the structure of intersubunit contact is formed by three sites: two identical peripheral sites, formed by loop 1–29 and helix 29–34 (H 29–34) of each subunit and one, located near to the active centers of two subunits. Destruction of two contacts does not effect the catalytic activity but opening the third results in the dissociation of dimers into monomers and loss of catalytic activity. Thus, kinetic calculation is correlated with the results of structural analysis. Oligomers with decreased activity and increased stability were found in solutions of E. coli AP. The structural possibilities for tetramer formation, based on packing of molecules in protein crystals, are discussed.