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

O.M. Poltorak (1), E.S. Chukhray (1), I.Y. Torshin (1), L.F. Atyaksheva (1), M.D. Trevan(2), M.F. Chaplin (3)
(1) Moscow State University, Chemistry Department
(2) Westminster University
(3) South Bank University, School of Applied Science

Archives of Biophysics and Biochemistry, 1998, in press

ABSTRACT. Alkaline phosphatase-phosphomonoester hydrolase (EC is a metalloenzyme that forms an homodimer. It hydrolyses small phosphomonoesters as well as the phosphate termini of DNA. Three-dimensional structural analysis of alkaline phosphatase (ALP, APase) from E.coli established that the structure of enzyme intersubunit contact is made by three contact sites: two identical peripheral sites, formed by loops 1-29 of each subunit and one central, located near to the active centres of two subunits. Destruction of these contacts results in the dissociation of dimers into monomers and the consequential loss of catalytic activity. Three separate contact areas are found between the dimeric subunits. When one, two or all three of these are destroyed under denaturing conditions in the external environment, labile dimeric isomers with different activity and stability are formed. Study of the alkaline phosphatases from various sources shows that the thermoinactivation curves, obtained under various conditions, have induction periods which 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 opening of the three contact sites that are making the conformational lock. Kinetic calculations show these stages to be interconvertible. In solutions of alkaline phosphatase, oligomers with decreased activity and increased stability can be formed. These oligomers contain more than two subunits and are supposedly tetramers. The structural possibilities for tetramer formation, based on hydrophilic and hydrophobic patch molecular surface analysis and molecular packing in protein crystals are discussed.

Laboratory of Kinetics and Catalysis