|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1995 : ¥200,000 (Direct Cost : ¥200,000)
Fiscal Year 1994 : ¥1,900,000 (Direct Cost : ¥1,900,000)
The zinc atom is an essential component of penicillolysin. Earlier studies found that the enzyme contains 1g-atom of zinc per mole of enzyme (Mr 17,000). The alpha-helix and beta-structure contents of the enzyme are ca 19 and 58%, respectively. The enzyme showed high affinity towards the Pro-X(X=Gln, Lys, Leu or Arg) bonds of substance P,dynorphine A,neurotensin and chicken brain pentapeptide, and the Arg-Arg bonds in dynorphine A and neuroteinsin. Preferential cleavages of bonds by the enzyme with hydrophobic amino acid residues at the P_1 position are observed on the peptide used. The specificity of penicillolysin differs from those of other metalloendopeptidase.
Site-directed mutagenesis of penicillolysin cDNAwas used to assess the functional role of catalytic residues. Our results demonstrate that a single amino acid substitution of Asn for Asp-121 or Asp-164 resulting in mutant enzymes causes drastic decrease in the catalysis of self-maturation with Zn^<2+> ions. The two mutants had no activity of clear zone formation. These results strongly support that Asp-121 and Asp-164 in penicillolysin are crucial for hydrolytic activity. The two essential aspartic acid residues differ from those of the thermolysin family. To identify the zinc ligands, we substituted Ala for His-128 or His-132. The result shows that His-128 and His-132 are essential amino acid residues and are identical to those of the zinc binding motif, HEXXH of the thermolysin family. Our result also shows that Glu-129 is an essential amino acid residue, while Glu-65 is not.
It is concluded that Asp-121, Asp-164 and Glu-129 are crucial for the hydrolytic activity of penicillolysin, that His-128 and His-132 are also crucial residues for zinc-coordinating enzymes, and that Asp-104 and Asp-143 may be binding sites of the enzyme towards basic substrate.