The Interplay of Catalytic Activity and Substrate Promiscuity for an Intrinsically Disordered Staphylococcal Nuclease

Abstract

In order to investigate the effect intrinsic disorder has on catalytic activity and enzyme-substrate promiscuity, we chose to study one N-, one C- and one N- and C-terminally truncated variant of Staphylococcal nuclease (SNase). SNase is a 149amino acid 3’ endo-exonuclease that is stable, refolds, and retains 75% of native activity in 5M Urea (Jackson and Chalkley, 1975). As SNase is well characterized biophysically and biochemically, comparative analyses of the effects of truncations on stability and catalytic activity, is feasible. By interfering with tertiary structure (N-terminal truncation), secondary structure (C-terminal truncation) or both, the degree of intrinsic disorder was altered, as measured by using circular dichroism, ANS fluorescence and 1D NMR. Following biophysical analyses, the catalytic activity on natural, and non-natural substrates was investigated. Using fluorescence spectroscopy with TAMRA and FAM labeled substrates, we found that the least structured variant; containing both N-terminal and C-terminal truncated regions, was the most active enzyme; exhibiting near native activity for the cleavage of an A16-mer. The order of catalytic efficiency for the substrates was similar between the variants. Nevertheless, introduction of phosphothioate linkages into the A16-mer - hypothesized to be nuclease resistant - was cleaved more efficiently than a C10-mer. No measurable activity was found for the two non-natural substrates chosen (TNA and pRNA). As intrinsic disorder is hypothesized to increase the number of protein sub-populations, with changed dynamics and kinetics as a consequence, bulk measurements are suboptimal to truly measure the actual catalytic efficiency. The results from this study serve as a good primer, enabling the choice of substrates with suitable turnover rates, for future single molecule FRET analyzes into intrinsically disordered SNase.