The effect of proteinases on phenylalanine ammonia-lyase from the yeast Rhodotorula glutinis

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Abstract
Phenylalanine ammonia-lyase of the yeast R. glutinis was rapidly inactivated by duodenal juice. It was susceptible to chymotrypsin and subtilisin and to a lesser extent trypsin. Initial proteolysis of the enzyme by chymotrypsin and trypsin resulted in cleavage of the monomeric subunit (75,000 MW) into a large (65,000 MW) and a small (10,000 MW) peptide. The small peptide was rapidly degraded. The 65,00 MW fragment was resistant to prolonged incubation with chymotrypsin, but was degraded by trypsin under the same conditions. Phenylalanine ammonia-lyase was cleaved into several polypeptides by subtilisin, the 65,000 MW peptide being totally absent. The N-terminal region of the enzyme was contained in the 65,000 MW fragment, as was the dehydroalanine moiety, the prosthetic group. Active-site-binding ligands protect the enzyme from inactivation by the 3 proteinases, and peptide-bond cleavage by trypsin and chymotrypsin. Several chemical modifications were performed on phenylalanine ammonia-lyase. Some decreased its antigenicity, and ethyl acetimidate decreased the rate of degradation of the 65,000 MW peptide by trypsin. The modification did not protect the enzyme from proteolytic inactivation of the enzyme activity. These observations are discussed in terms of the structure of phenylalanine ammonia-lyase and site of action of the proteinases.