David Coffman; Carson Cole, Weber State University
Proteases are a class of enzymes whose function is to cleave peptides into smaller amino acid chains or free amino acids. Two such proteases, Acyl Peptide Enzyme Hydrolase (APEH) and acylase (ACY), function in unison to cleave N-acylated amino acids from the N-terminal end of peptides so that these peptides can become further metabolized in the cell. In this pathway, APEH will first cleave an N-acylated amino acid from the end of a peptide. Then, ACY will remove the acetyl group from the amino acid so it can be recycled or further metabolized. While the enzymatic activity of APEH and ACY have been studied extensively alone, little has been done to study flux through the pathway. Understanding enzyme flux is useful since enzymes rarely work independently of one another in vivo but rather work in a coordinated fashion to perform their respective functions. This research illustrates a novel method to observe how both enzymes work together using Gas Chromatography Mass Spectrometry (GCMS). Using a selected N-acetylated dipeptide, the release of 2 free amino acids-Ala and Met- is detected by GC-MS and correlates to APEH and ACY activity, respectively. Using standard curves and time courses, the rate through APEH and ACY was determined. This method serves as a relatively simple way to study enzymes in vitro in an attempt to better understand the pathways function in vivo. In addition to measuring the flux of more than one enzyme in metabolic pathway, the use of this GCMS method has revealed that the enzymatic activity of the proteases in question do not function universally across different cell lysates. For example, it was determined that the rate of flux through APEH/ACY was 5 times faster in A549 cancer cell lysates than in normal blood lysates. The GCMS method described herein allows comparative studies of APEH/ACY flux and has potential to deepen the knowledge of how other these enzymes work together in both normal and disease states.