Studies of the enzyme phosphoenolpyruvate (PEP) carboxylase from maize will be conducted to map the residues involved in the binding the inhibitor malate, and to probe the physical basis of the interaction between allosteric effectors and regulatory phosphorylation. Conditions for preparing diffraction-quality crystals of the phospho and dephospho forms of the enzymes will be sought. Unlike the bacterial form of the enzyme, which effects of phosphorylation at Ser15 and several allosteric effectors. Phosphorylation is not required for form of the enzyme is less sensitive to inhibition by malate and more sensitive to the activator glucose-6 phosphate. Based on recent findings on the physical basis for interaction between allosteric regulation and reversible phosphorylation in yeast glycogen phosphorylase (Lin et al. 1996), and the recently published competitive interaction between the phosphorylated serine and the inhibitor at the allosteric inhibition site. The model predicts that the allosteric effectors of PEP carboxylase modulate the enzyme's susceptibility to dephosphorylation by protein phosphatase. We will test this model by monitoring phosphatase activity in vitro using 33P-labeled recombinant maize PEP carboxylase and type 2A protein phosphatase. The release of 33PO4 from the phosphorylated enzyme will be followed in the presence and absence of allosteric inhibitors and activators and over a range of pH known to modulate allosteric behavior. Site-directed mutagenesis will be used to probe the functional role of various residues in regions of the enzyme that appear to be involved in inhibitor binding. The results are expected to provide insight into the interplay of covalent and non-covalent regulatory mechanisms of enzymes in general, and specific introduction about the inhibitor response of this enzyme which, though it is not found in higher animals, plays a key role in bacteria, plants and parasitic organisms.

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California State University Los Angeles
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