The Sequence around the Active‐Center Tyrosyl of Porcine Carboxypeptidase B

Iodination of porcine carboxypeptidase B causes a loss of both peptidase and esterase activity; the presence of the inhibitor β‐phenylpropionate during iodination prevents the loss. A residue in the active site of the enzyme was labeled by a procedure in which one portion of the enzyme was iodinated with 125 I‐labeled iodine and a second portion with 131 I‐labeled iodine in the presence of the inhibitor β‐phenylpropionate. The two preparations were mixed, digested with elastase and the iodopeptides separated. Two iodopeptides which appear to have come from the active site were isolated; both had high 125 I/ 131 I radioactivity ratios relative to the unfractionated digest. The labeled residue is tyrosyl in both iodopeptides. The same results were obtained when the inhibitor ɛ‐aminocaproate was used instead of β‐phenylpropionate. The yield of the two iodopeptides approached a total of about 1 mole/mole of enzyme in each of several experiments. The sequences of the two iodopeptides were found to be Thr‐Ile‐(monoiodo)Tyr‐Pro‐Ala and Ile‐(monoiodo)Tyr‐Pro‐Ala, and both appear to represent the same tyrosyl residue in the site of porcine carboxypeptidase B.