Carbamylation of Pepsinogen and Pepsin

Abstract 1. Potassium cyanate, a reagent that carbamylates proteins, reacts both with pepsinogen and pepsin. 2. When allowed to react with pepsinogen, nine of the 10 e-NH2 groups of the lysine residues are carbamylated and homocitrulline is formed. In addition, reaction occurs with 4 tyrosine residues but not with the α-NH2-terminal leucine. 3. Carbamylation of pepsinogen is accompanied by a decreased susceptibility to activation. The enzymically active product that can be obtained is, in its amino acid composition and NH2-terminal amino acid, indistinguishable from pepsin prepared from the untreated zymogen. 4. Carbamylation results in a change of the molecular conformation of the protein as reflected by a decrease of λc from 236 to 220 mµ, an increase of the levorotation, [α]366, from -212 to -297, and a change of [m'] from -4086 to -3802. 5. Pepsinogen in which 3 lysines are carbamylated retains its native conformation and full potential pepsin activity. On further reaction with potassium cyanate, the loss of potential pepsin activity parallels the percentage change of the specific optical rotation, [α]366. 6. When pepsin is carbamylated, the proteolytic activity of the enzyme toward hemoglobin decreases, whereas the hydrolysis of the synthetic substrate, N-acetyl-dl-phenylalanyldiiodotyrosine, increases 2.8-fold. These changes can be reversed by decarbamylation with hydroxylamine. 7. With the aid of differences in the absorbance and in the incorporation of K14CNO it was established that 6 tyrosine residues were carbamylated in pepsin. Although the NH2-terminal isoleucine had reacted, the 1 lysine residue of the protein was not carbamylated.