Due to the stability of the substrate-enzyme complex created by covalent nucleophilic attack, it is observed that the rate determining step of E. coli alkaline phosphatase (EcAP) kinetics is dependent on the release of the dephosphorylated product. In the evaluation of the structural stability of EcAP, highly stabilizing intramolecular interactions are present, specifically disulfide bonds at the C168-C178 and C286-C336 junctions. This research performed single point mutations at both C168 and C178 positions to alanine, and finally a double point mutation at both residues to give A168-A178. These mutants were subject to kinetic studies using typical substrates (p-nitrophenyl phosphate) as well as substrates with low promiscuous activity, similar functional groups (sulfate and phospho-monomethyl-ester). All mutants were furthermore subject to tungstate inhibition for clear determination of Km values. Results were twofold: first, mutants demonstrated significantly reduced Km values and greatly increased catalytic efficiency when compared to wild type; and second, reduction in stabilizing intramolecular interactions distal to the dimer interface and proximal to the active site allowed for promiscuous behavior. This data indicates that protein architecture is intimately linked with both catalytic efficiency and promiscuity, areas of protein kinetics which are rapidly developing.

Disulfide bridge mass detruction to induce catalytic promiscuity

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