Abstract 1856 Investigating Allosteric Regulation of Glucan Phosphatase Starch Excess4

Glucan phosphatase Starch Excess4 (SEX4) is an enzyme essential for starch degradation via reversible phosphorylation. Previous studies of SEX4 orthologs display a unique sigmoidal kinetic response to soluble amylopectin characterized by a Hill coefficient > 1.0, suggesting a signature feature of cooperativity. It is still unclear whether the deviation from Michaelis-Menten hyperbolic dependence is due to the presence of more than one binding site or simultaneous interactions with several substrates and/or allosteric modifiers. We explored the structural basis for positive kinetic cooperativity using SEX4 from the model plant Arabidopsis thaliana and agronomically important Zea mays. Using site-directed mutagenesis, we generated and expressed SEX4 mutant proteins in E.coli and assessed their catalytic activity against soluble and insoluble starch substrates. Our findings suggest that residues directly interacting with the SEX4 carbohydrate-binding module (CBM) dramatically influence the binding cooperativity and substrate transformation rates. Future studies will be focused on identifying the possible allosteric modulators for the SEX4 activity and characterizing enzymatic activity against various glucan substrates. The research was supported by National Science Foundation (NSF- NCB2012074)