Feasibility of Using Saliva as a Biospecimen for Breast Cancer Screening in Women

Serine proteases play an important role in the regulation of most biological processes. Malfunctions in this class of enzyme lead to a variety of diseases and thus are an important target in medicinal chemistry. Trypsin is a digestive proteinase which cleaves peptide bonds at the carboxyl side of lysine or arginine via the conserved catalytic triad (Ser 195, His 57, and Asp 102) which is characteristic of serine proteases. It also contains a highly conserved disulfide bridge (C42 – C58) near the catalytic triad. Previously, by using enzymatic assays, it has been shown that replacing serine with threonine (S195T variant) results in a significant loss of activity. However, it was found that removing the nearby disulfide bridge in the S195T variant (C42A C58A S195T; AA-Th variant) returns activity to more than half that of wild type. In our work using AMBER, a molecular dynamics simulation package, we provided structural rationale to the differences in enzymatic activity for two variants (S195T and AA-Th) as compared to wild type. >From our results, the key component to the activity of the protease was the conformation of the hydroxly group of threonine. Steric constraint imposed by the disulfide bridge prevented the hydroxly group from occupying the area necessary for catalysis. In addition, the methyl moiety of T195 should interfere with the carbonyl group of an incoming substrate. This conformation of the hydroxly group in the S195T variant was consistently different to wild type and the AA-Th variant. In the AA-Th variant, as observed by the long range MD simulations, the lack of the disulfide bridge increased the conformational flexibility of threonine residue and allowed the hydroxly group to be in the catalytically active orientation.