Many proteins are functionally regulated by the O-GlcNAc post-translational modification, and O-GlcNAcase (OGA) is the only cytosolic enzyme known to remove this sugar. We are proposing a new method to identify the O-GlcNAc modification could be based on a mutant OGA enzyme that binds to O-GlcNAcylated proteins without cleaving the modification or releasing the substrate. Furthermore, studying mutations in the OGA active site can lead to a better understanding of the enzyme’s structure-function correlation. Two adjacent aspartic acids, D174 and D175, comprise OGA’s active site, while amino acids G67, K98, N280, D285, N313 are included in its binding site. An in silico model of the wild-type (WT) OGA was used to study the enzyme-substrate interaction and was represented by a homology model provided by de Alencar et al. UCSF Chimera visualization software and a molecular dynamics package were used to prepare WT and mutated OGA structures. AutoDock was used to run binding simulations on these structures with the substrate PUGNAc, a competitive inhibitor. These computational results directed the decision on which OGA mutations to experimentally investigate. OGA enzymes were expressed in E. coli bacteria and purified by metal-affinity chromatography. Enzyme assays using a para-nitrophenol-GlcNAc (pNP-GlcNAc) substrate were used to investigate OGA’s activity, with the extent of the enzymatic reaction quantified by spectroscopy. Isothermal titration calorimetry (ITC) was used to study the binding activity of OGA. These experimental results will be compared to the computational data in order to evaluate the accuracy of the homology model and docking studies. The goal is to identify OGA mutants that can bind substrate without cleaving or releasing it.

Computational and experimental approaches to investigate substrate binding of the enzyme OGA

249th National Meeting (2015)

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