STRUCT & FUNCT OF MUTANT VERSIONS OF ALKALINE PHOSPHATASE FROM ECOLI

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The goals of this project are to acquire a deeper understanding of the relationship between protein structure and function by using alkaline phosphatase as a model system. The function of this enzyme is to catalyze the nonspecific hydrolysis of phosphate esters, and the lack of the activity of this enzyme results in the fatal hereditary disease hypophosphatasia. Alkaline phosphatase from mammals is closely related to the corresponding bacterial enzyme, and the enzyme from Escherichia coli has become the model for the study of all alkaline phosphatases. Additional time on the Y-MP C90 at PSC is being requested in order to carry on with this project. This involves using the program XPLOR to refine the x-ray crystallography data on mutant versions of the enzyme for which data is already available, and at least four others for which data will be collected shortly. We will also use of XPLOR and CHARMM to carry out molecular dynamics calculations on mutant versions of the enzyme in order to help understand their unusual properties. Local computer facilities are not capable of carrying out these calculations in reasonable times. Correlations will then be made between the functional changes induced by the amino acid substitution and the three-dimensional structure of the mutant enzymes. This work will not only be important for the understanding of this particular system, but more importantly for formulating general concepts about enzyme catalysis, and the function of metals in proteins, and providing a molecular explanation for intergenic complementation.