Phosphoenolpyruvate (PEP) carboxylase is of interest because of its central role in the photosynthetic metabolism of CAM and C4 plants. As would be expected for a key enzyme in a major metabolic pathway, the activity of the enzyme is highly regulated, and a fundamental regulatory element appears to be the interplay of the activation caused by phosphorylated activators such as glucose 6-phosphate and AMP and the inhibition caused by malate and ATP. The overall goal of this research is to elucidate various molecular aspects of the process by which effectors, particularly activators such as AMP and glucose 6-phosphate, regulate PEP carboxylase from Crassula argentea and other sources. The research will probe the binding site(s) of activators and inhibitors through the use of photoaffinity labels. The proposed research will also test the molecular mechanism of activation, in light of a novel hypothesis that has recently been proposed.
The specific aims i nclude estimating the number of activator binding sites and the dissociation constants for the activators, determining whether all activators share a common binding site, comparing the AMP and ATP binding sites, identifying the peptide sequence at the activator binding site, and testing the role of activator dephosphorylation in the activation process. The results are expected to provide molecular information on how and where activators exert their influence on the enzyme. This information will provide greater insight into the structure-function relationships of this specific enzyme and, more generally, into the process of metabolic regulation at the enzyme level.
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