Carboxypeptidase I from triticale grains and the hydrolysis of salt-soluble fractions of storage proteins.

Carboxypeptidase I was purified from triticale grains (×Triticosecale Wittm.) by a 5-step purification procedure including gel filtration, cation-exchange chromatography and affinity chromatography. The enzyme was purified 595.9 fold with a 1.58% recovery. Triticale carboxypeptidase I is a homodimer with a molecular weight of 124.2 kDa and a subunit weight of 55.2 kDa. Each subunit is composed of two polypeptide chains (33.4 and 21.3 kDa). Serine was found in the active site of triticale carboxypeptidase I; DFP (diisopropylflourophosphate) and other applied inhibitors of serine proteases inhibited the enzyme activity. Triticale carboxypeptidase I hydrolyzes N-CBZ-dipeptide (N-carbobenzoxy-dipeptide) substrates at low pH. N-CBZ-Phe-Ala, N-CBZ-Phe-Leu and N-CBZ-Ala-Met were hydrolyzed with the highest rates. The lowest K(m) value and the highest k(cat)/K(m) ratio were observed for hydrolysis of N-CBZ-Phe-Ala. Studies on the amino acid sequence revealed that the purified enzyme is homologous to carboxypeptidase I from barley. Analyses of conserved regions in the sequence of triticale carboxypeptidase I revealed the presence of Ser, Asp and His that compose the catalytic triad. Intact storage proteins were poor substrates for carboxypeptidases. Carboxypeptidase I together with carboxypeptidase III effectively degraded albumins proteolytically modified by endopeptidase EP8. Modified globulins were degraded at a slower rate, and all three carboxypeptidases were required for a significantly increased activity. Studies of the expression of the carboxypeptidase I gene revealed that the synthesis of the enzyme occurs mainly in the scutellum of the grain. The enzyme is also expressed in the aleurone layer of the grains, although its function in this tissue is unknown.