This proposal is to study the regulatory mechanism of 3- phosphoglycerate dehydrogenase, an enzyme that is the committed step in de novo serine biosynthesis. Serine is a negative allosteric inhibitor. The crystal structure indicates that the regulatory site is in a domain separated from other two domains, the nucleotide and substrate binding domains. The hypothesis is that a domain motion of the nucleotide and substrate binding domains has to occur for catalysis to take place. Binding of serine at the regulatory site prevents this domain motion. Further, the binding of serine at the regulatory site increases the interaction between two regulatory sites. Preliminary experiments indicate that cross-linking the adjacent regulatory domains results in inhibition, whereas site directed mutagenesis experiments weakening the interaction between subunits prevents inhibition by serine. This proposal uses a variety of fluorescence methodologies and continued crystallographic efforts to determine the mechanism of allosteric control.
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