Probing Protease Active Sites with Vibrational Reporters

Abstract

The environment of the active site of an enzyme greatly influences its activity. Here, analogues of arylsulfonyl fluoride enzyme inhibitors were modified with either a nitrile or azide vibrational reporter to inhibit and probe the active site of several proteases including subtilisin and trypsin. Nitrile and azide groups are effective vibrational reporters due the size of the reporters and the position and sensitivity of the nitrile symmetric and azide asymmetric stretch frequency, respectively. The sensitivity of the nitrile and azide stretch of the modified inhibitors were first measured in a range of solvents designed to mimic various biological environments to assess the sensitivity of these observables to local environment. This initial work permitted the observed nitrile and azide stretch frequencies of the inhibitors bound to the proteins to be correlated to the local hydration state of the active sites. These infrared spectroscopic results of the inhibitor and protein-inhibitor complexes will be presented in addition to the synthesis of the modified inhibitors and relevant protein crystal structures of these protein-inhibitor complexes.