Studies on aspartase. VI. Trypsin-mediated activation releasing carboxy-terminal peptides.
Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) of Escherichia coli, already of full activity, is 3-5-fold activated by a limited proteolysis with trypsin (Mizuta, K. and Tokushige, M. (1976) Biochim. Biophys. Acta 452, 253-261). Structural bases for the activation were investigated. The NH2-termini of the native enzyme and of the trypsin-activated enzyme were found to be equally serine, as analyzed by the dansylation method. However, the COOH-terminal glutamate of the native enzyme was altered to arginine upon activation, as revealed by treatments with carboxypeptidases Y, A and B. The released peptides were obtained by molecular sieve membrane filtration following trypsin activation of the enzyme. The peptides were separated by high voltage paper electrophoresis, and the amino acid composition and the terminal residues were determined. The results showed that one or a few related peptides consisting of 7-17 residues were released from the COOH-terminal upon activation. The circular dichroism spectrum of the enzyme suggested that the helical content of the activated enzyme was about 5% less than that of the native enzyme, an indication that the trypsin-activated enzyme has a somewhat looser conformation than the native enzyme. Determination of the fluorescence decay time of the enzyme protein indicated that the tryptophan residue became more exposed to outer environment than that of the native enzyme upon trypsin-activation.
D000596:Amino Acids / Q000032:analysis; D000642:Ammonia-Lyases / Q000378:metabolism*; D001220:Aspartate Ammonia-Lyase / Q000378:metabolism*; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000494:pharmacology*
D000596:Amino Acids; D010446:Peptide Fragments; D014357:Trypsin; D000642:Ammonia-Lyases; D001220:Aspartate Ammonia-Lyase