Distal Residues and Enzyme Activity: Implications for Personalized Medicine

Abstract

Phosphorylation regulates protein conformation and function, and in turn, is regulated by phosphatases which rapidly dephosphorylate tyrosine, serine, and threonine residues through the hydrolysis of phosphomonoester bonds. The turnover number for phosphatases is generally high. For example, a single molecule of tissue non-specific alkaline phosphatase can catalyze nearly 1000 dephosphorylations per second. Therefore, residual phosphatase activity in a sample risks the omission of important phosphorylation events from the analysis. High pressure has been shown to enhance the proteolytic digestion of proteins preceding mass spectrometry, resulting in significantly reduced digestion times, fewer miscleavages, and improved sequence coverage. Lysyl endopeptidase (Lys-C) is commonly used for proteins resistant to trypsin digestion or may be used preemptively in combination with trypsin. The resiliency of Lys-C is clearly demonstrated where the rate of substrate conversion is accelerated by an order of magnitude at elevated pressure and temperature. In these experiments, high pressure optical spectroscopy (HiPOS) was used to measure in real time the activity of alkaline phosphatase (ALP). ALP is reversibly inactivated at high pressure and partial activity may be restored upon return to atmospheric pressure. HiPOS demonstrated the bufferdependent restoration of ALP activity following pressurization to 45 kpsi and return to atmospheric making the method a useful monitor of protein conformational dynamics. At the elevated temperature and chaotrope concentration typically used for proteolytic digestion, the inactivation of ALP by Lys-C was marginal at atmospheric pressure providing an opportunity for extensive artifactual dephosphorylation, whereas at high pressure, ALP is rapidly inactivated.