A variety of diseases including sickle cell anemia, beta- thalassemia, Tay-Sachs and phenylketonuria are the result of a single amino acid alteration in the structure of a particular protein or enzyme. Although we can determine the structure of a protein to atomic resolution, we still do not understand how, in detail, this structure is related to the function of the particular protein of enzyme. In order to understand the molecular basis of diseases, we need to elucidate at the molecular level the relationship between protein structure and function. Therefore, the long term goals of this project are to acquire a deeper understanding of the relationship between protein structure and function by using E. coli alkaline phosphatase as a model system. This enzyme catalyzes the nonspecific hydrolysis of phosphate esters, and is the model for the study of all alkaline phosphatases.
The specific aims of this proposal are to answer fundamental questions concerning the relationship between structure and function of alkaline phosphatase. We will concentrate on the molecular details of the catalytic mechanism, the mode by which information is passed between the subunits of the enzyme, and the function of the metals in this enzyme. We will use a variety of molecular biology techniques to create altered versions of the enzyme with single amino acid substitutions. Initially, work will concentrate on the analysis of mutants that have been already created. Selection of additional sites for amino acid substitutions will be based on all the biochemical and structural data currently available. Kinetic and biophysical methods such as stopped-flow kinetics, circular dichroism, NMR spectroscopy, and X-ray crystallography will be used to analyze the results of the amino acid substitutions. Correlations will 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, cooperativity and the function of metals in proteins.
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