A truncated form of human procaspase-9 missing the first 111 amino acids, and a variety of mutants derived therefrom, have been expressed in Escherichia coli inclusion bodies. Upon refolding to active enzymes, Delta(1-111) procaspase-9 and mutants were recovered at purity greater than 95% and with a final yield of 20-35 mg/L cell culture. Our active procaspase-9 retains its pro-seginent, while undergoing major auto processing Lit Asp(315) and a minor (20%) cleavage at Glu(306). This unusual cleavage at a Glu-X bond also took place in the D315E Mutant, and we describe herein the inhibitor Z-VAE-fmk that shows enhanced inactivation of procaspase-9 over caspases-3. The bond at Asp(330), not processed by procaspase-9, is cleaved by caspase-3 and the resulting procaspase-9 variant, missing the 316-330 bridge, is six times as active as the non-mutated A(1-111) proenzyme. A deletion mutant lacking residues 316-330 underwent auto activation by cleavage at Asp(315)-Ala(331) bond. Moreover, substitution of Glu(306) by an Asp residue in this mutant led to rapid removal of the peptide spanning Ser(307) to Asp(330), and resulted in an enzyme that was 7.6 times as active as the non-mutated A(1-111) procaspase-9. Finally, replacing both Asp(315) and Glu(306) with Ala generated a procaspase-9 mutant incapable of auto processing. This single chain procaspase-9 was fully as active as the non-mutated Delta(1-111) enzyme processed at Asp(315) or Glu(306). Our demonstration that Unprocessed procaspase-9 Mutants are active as proteases with caspase-type specificity suggests that the role of procaspase-9 in cascade activation of executioner caspases might, in some circumstances, be carried out alone and without association of the apoptosome. (c) 2005 Elsevier Inc. All rights reserved.