Modification of pepsinogen with pyridoxal phosphate.

Pyridoxal-PO* binds to pepsinogen in a reaction in which the stoichiometry is highly dependent on the conformation of the protein. When pepsinogen is in its native conformation, pyridoxal-PO4 forms Schiff bases with the (r-NH2 group of Leul and the e-NH2 group of LysZS8. When the protein is mildly denatured and assumes a more extended conformation, pyridoxal-PO4 can react with the t-NH2 groups of approximately 3 other lysine residues present in the NHzterminal region of the pepsinogen molecule. Pepsinogen with pyridoxal-PO4 covalently attached at Leul and Lysri~ by reduction of the protein-pyridoxal-PO4 Schiff bases can be activated to pepsin. This modified pepsin, which has a pyridoxal-PO., molecule covalently attached to the e-NH2 group of its sole lysine residue, cannot be distinguished catalytically from the unmodified enzyme. As soon as 1 or 2 of the lysine residues in the NH*-terminal region of pepsinogen become modified with pyridoxal-Pod, the zymogen can no longer be activated, possibly because the protein cannot assume the proper conformation required for activation. No pattern to the sequence of pyridoxal-PO4 incorporation into pepsinogen was observed after the first 2 pyridoxal-PO4 molecules were incorporated. The lysine residues showing the greatest reactivity toward pyridoxal-PO4 other than LYS:.~~ were found in regions of the NH2 terminus in which the density of basic amino acids was highest.