Characterization of the interactions between the small GTPase RhoA and its guanine nucleotide exchange factors.

A novel spectrophotometric method to study the kinetics of the guanine nucleotide exchange factors-catalyzed reactions is presented. The method incorporates two coupling enzyme systems: (a). GTPase-activating protein which stimulates the intrinsic GTP hydrolysis reaction of small GTPases and (b). purine nucleotide phosphorylase and its chromophoric substrate, 7-methyl-6-thioguanosine, for quantitation of the resultant inorganic phosphate. The continuous coupled enzyme system was used for characterization of the interactions between the small GTPase RhoA and its guanine nucleotide exchange factors, Lbc and Dbl. Kinetic parameters obtained here show that there is no significant difference in kinetic mechanism of these GEFs in interaction with RhoA. The Michaelis-Menten constants were determined to be around 1micro M, and the rate constants k(cat) were around 0.1s(-1).