STRUCTURAL STUDIES OF THE ACTIVE SITE OF ALKALINE PHOSPHATASE : FROM BACTERIAL TO HUMAN
M. H. le Du, J. B. Charbonnier, J. C. Boulain, E. A. Stura, & A. Ménez.
Département díingénierie
et díétudes des protéines, CEA Saclay, 91191 Gif sur Yvette
Cedex
email : ledu@balthazar.saclay.cea.fr
Alkaline phosphatases are dimeric metalloenzymes which catalyse the hydrolysis of phosphomonoesters. A reaction mechanism has been proposed on the basis of the crystallographic structure (1) and kinetic data. The reaction proceeds through a pentaedral phosphoseryl intermediate (2).
Bacterial and mammalian alkaline phosphatase share 25-30% identity, but functional divergences have been observed : bacterial alkaline phosphatase are more thermostable than mammalian enzymes while mammalian enzymes have a catalytic activity 20 to 30 times higher than the bacterial enzymes, an optimal pH of 10, and are activated by magnesium.
We studied a double point mutant D153H/D330N of the E. Coli alkaline phosphatase which has gained through this mutation mammalian biochemical properties : a 17 time higher kcat than the wild type enzyme, an optimal pH of 10, and a magnesium dependant activity. Each correspondant single point mutant shows an increase of the kcat of 3-5 fold, consistent with a synergistic effect of the double point mutation.
Various structures have been solved by molecular replacement : with and without magnesium, with and without phosphate in the active site, and with and without aluminium fluoride, a mimic of the pentahedral reaction intermediate. The different states of this mutant will be compared to the structure of the wild type enzyme (1), and the single point mutants D153H (3) and D300N.
We also solved the structure of the human placental alkaline phosphatase by a combination of molecular replacement and isomorphous replacement. The rebuilding of the structure is well advanced.