Specificity of yeast KEX2 protease for variant human proalbumins is identical to the in vivo specificity of the hepatic proalbumin convertase.

Yeast KEX2 protease was examined as a potential model for a human proprotein convertase and, in all respects, mimicked the predicted properties of a proalbumin convertase. The enzyme rapidly cleaved the propeptide Arg-Gly-Val-Phe-Arg-Arg from the NH2-terminal end of proalbumin but, unlike trypsin, failed to cleave physiologically unprocessed human proalbumin variants. There was little or no cleavage of proalbumin Lille (Arg-2----His) or Christchurch (Arg-1----Gln), and there was negligible cleavage of proalbumin Blenheim (Asp1----Val), despite the fact that it retains the dibasic processing signal. Proalbumin Kaikoura (Arg-2----Cys), which appears to be partially processed in vivo, was cleaved at about half the rate of normal proalbumin despite the absence of a diarginyl sequence. Restoration of a dibasic site through aminoethylation of the new cysteine increased the rate of cleavage to near that of normal proalbumin. The KEX2-catalyzed cleavage of normal proalbumin was found to be independent of pH between pH 6.0 and 8.0. Antitrypsin Pittsburgh (Met358----Arg), a predicted specific inhibitor of in vivo proalbumin cleavage, inhibited KEX2 in a reversible manner. A molar excess of thrombin over antitrypsin Pittsburgh relieved the inhibition of KEX2, suggesting that a covalent complex is not formed between KEX2 and the inhibitor.