Refined Crystal Structure of the Potato Inhibitor Complex of Carboxypeptidase A at 2.5 Å Resolution

Abstract

The exopeptidase carboxypeptidase A forms a tight complex with a 39 residue inhibitor protein from potatoes. We have determined the crystal structure of this complex, and refined the atomic model to a crystallographic R-factor of 0.196 at 2.5 Å resolution. The structure of the inhibitor protein is organized around a core of disulfide bridges. No α-helices or β-sheets are present in this protein, although there is one turn of 3_(10) helix. The four carboxy-terminal residues of the inhibitor protein bind in the active site groove of carboxypeptidase A, defining binding subsites S′_1, S_1, S_2 and S_3 on the enzyme. The carboxy-terminal glycine of the inhibitor is cleaved from the remainder of the inhibitor in the complex, and remains trapped in the back of the active site pocket. Interactions between the inhibitor and residues Tyr248 and Arg71 of carboxypeptidase A resemble possible features of binding stages for substrates both prior and subsequent to peptide bond hydrolysis. Not all of these interactions would be available to different types of ester substrates, however, which may be in part responsible for the observed kinetic differences in hydrolysis between peptides and various classes of esters. With the exception of residues involved in the binding of the inhibitor protein (such as Tyr248), the structure of carboxypeptidase A as determined in the inhibitor complex is quite similar to the structure of the unliganded enzyme (Lipscomb et al., 1968), which was solved from an unrelated crystal form.

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