Carboxypeptidase inhibitor from potatoes. The effects of chemical modifications on inhibitory activity

Abstract

The carboxypeptidase inhibitor from Russet Burbank potatoes was subjected to a variety of chemical modifications and their effects on inhibitory activity toward carboxypeptidases A and B were determined. The importance of the alpha carboxylate of glycine-39 to the enzyme-inhibitor interaction was demonstrated by the observation that a derivative in which all four carboxyls were modified was inactive whereas a derivative in which only the beta carboxylates of aspartic acid residues 5, 16, and 17 were masked retained full inhibitory activity. In addition to these three aspartic acid residues, lysine residues 10 and 13, histidine residues 3 and 15, and arginine-32 were modified and residues 1-5 removed with little effect on inhibitory activity. Tryptophan residues 22 and 28 did not react with 2-hydroxy-5-nitrobenzyl bromide or o-nitrophenylsulfenyl chloride, and thus are presumed to be buried in the interior of the inhibitor molecule. Although tyrosine-37 was acetylated without affecting binding characteristics, both carboxypeptidases A and B protected against deacetylation by hydroxylamine. These studies indicate that the carboxyl terminal region of the inhibitor is in contact with enzyme in the complex. The parallel effects of modifications on inhibitory activity toward carboxypeptidases A and B support previous evidence that both enzymes utilize the same binding site on the inhibitor [C. A. Ryan (1971), Biochem. Biophys. Res. Commun. 44, 1265].

Structure of the potato inhibitor complex of carboxypeptidase A at 2.5-A resolution.

“…Consistent with the CPase A-PCI complex structure, chemical modification studies on PCI had predicted the involvement of Gly-39 and Tyr-37 and a smaller role for His-15 in inhibitor binding to CPase A (27). Likewise, the crystal structure confirms the lack of involvement of Lys, Arg, and Asp side chains, and the amino-terminal five amino acids, in the binding to CPase A.…”

Rat peritoneal mast cell carboxypeptidase: localization, purification, and enzymatic properties

“…Sprague-Dawley rats were obtained from Tyler Labs (Bellevue, WA), Potato carboxypeptida~ inhibitor [6] and carboxypeptidase inhibitor-agarose [7] were generous gifts of Dr G. Michael Hass.…”

Role of proteolytic enzymes in biological regulation (a review).

“…In fact, only a few residues of the inhibitor molecule interact with the active site of the enzyme, whereas the bulk of the inhibitor serves mainly the function of a supporting structure. This is true of the pancreatic and soybean trypsin inhibitors, as well as of the carboxypeptidase inhibitor from potatoes that has been recently investigated in our laboratory (24). Cascades of zymogen activation reactions do not necessarily operate in isolation but may influence one another in the initial stages, by positive or negative feedback regulation, thus adding another element of control.…”