Faculty Research Grant 2022-2023

Peter Lyons (Biology).

Activation mechanisms for novel carboxypeptidase enzymes.

Abstract: The structure and function of a protein is determined by its sequence of amino acids. Protease enzymes can cut these amino acid sequences, leading to activation or inhibition of protein function. Because the action of proteases is irreversible, their activity must be tightly controlled so that they act only when needed. Proteases are commonly activated through the removal of an inhibitory prodomain or through the interaction of an activating allosteric molecule. We aim to investigate the activation mechanisms of two protease enzymes. First, we will explore the activation of carboxypeptidase O (CPO) by the allosteric interaction of glutamate-extended folate. The CPO protein will be hexahistidine tagged, expressed in Sf9 insect cells, and purified for subsequent enzymatic analyses in the presence or absence of various folate molecules. Secondly, a study of gene duplication in the metallocarboxypeptidase family requires the analysis of the activity of four such enzymes found in Xenopus frogs. The inhibitory prodomains of these enzymes will be removed through either enzymatic or bioengineering methods, followed by examination of activity. Finally, a protease enzyme from Agaricus bisporus suggests a unique catalytic mechanism. However, the removal of the prodomain is necessary for reliable analysis of enzymatic activity. This will be performed as for Xenopus CPO. Altogether, three independent projects will be advanced using similar approaches to solve a common problem: activation of an enzyme necessary for further study of its biochemical and biological function. All three projects will involve undergraduate students and will result in publication in the coming years.