The effect of proteinases on phenylalanine ammonia-lyase from the yeast Rhodotorula glutinis.
Phenylalanine ammonia-lyase (EC 4.3.1.5) of the yeast Rhodotorula 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 Mr) into a large (65 000 Mr) and a small (10 000 Mr) peptide. The small peptide was rapidly degraded. The 65 000-Mr 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-Mr peptide being totally absent. The N-terminal region of the enzyme was contained in the 65 000-Mr fragment, as was the dehydroalanine moiety, the prosthetic group. Active-site-binding ligands protect the enzyme from inactivation by the three 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-Mr peptide by trypsin. The modification did not protect the enzyme from proteolytic inactivation of the enzymic activity. These observations are discussed in terms of the structure of phenylalanine ammonia-lyase and site of action of the proteinases.
D000642:Ammonia-Lyases / Q000037:antagonists & inhibitors*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008024:Ligands / Q000494:pharmacology; D003904:Mitosporic Fungi / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases / Q000494:pharmacology*; D010650:Phenylalanine Ammonia-Lyase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012248:Rhodotorula / Q000187:drug effects / Q000201:enzymology*
D008024:Ligands; D010446:Peptide Fragments; D010447:Peptide Hydrolases; D000642:Ammonia-Lyases; D010650:Phenylalanine Ammonia-Lyase