Computational Studies of Selected Mutants of Shrimp Alkaline Phosphatase

Abstract

Drug discovery and refinement for use in the industry is an important and lucrative field within chemistry. In this regard, computational chemistry has shown to be a valuable tool in assisting drug discovery. A set of intriguing proteins used in drug discovery are cold-adapted enzymes. These enzymes have a lower activation point which makes them of interest in industrial use. In this study, the cold-adapted shrimp alkaline phosphatase’s (SAP) affinity towards the immunoassay substrates PNPP and AMPPD has been studied. With free energy calculations using the linear interaction energy (LIE) and free energy perturbation (FEP) methods, SAP’s affinity towards the substrates has been determined. In addition, putative mutations of SAP have been carried out by modelling SAP upon the already catalytic effective calf intestinal alkaline phosphatase (CIP) to increase its affinity towards PNPP and AMPPD. It was found that both substrates bound favourably to SAP and all its different mutations. Specificity towards PNPP and AMPPD changed depending on the mutations, where G102R would bind PNPP over AMPPD, while H149D would bind AMPPD over PNPP. The most successful mutation was a metal exchange of Zn2+ in the M3 metal site to Mg2+ according to the LIE method. The FEP method on the other hand revealed a decrease in binding free energies when transforming Zn2+ to Mg2+. One possible solution given is that a key water molecule coordinated poorly in the FEP simulations compared to LIE simulations. However, even though literature shows that Mg2+ increases the catalytic effect, this does not necessarily imply that the proteins affinity towards the substrates need to increase.