THERMITASE - A THERMOSTABLE SERINE PROTEASE .4. KINETIC INVESTIGATIONS ON THE BINDING OF N-ACYLATED PEPTIDE KETONES AS SUBSTRATE ANALOGOUS INHIBITORS

  • 1 January 1984
    • journal article
    • research article
    • Vol. 43  (7) , 887-899
Abstract
Peptide methyl ketones are efficient competitive inhibitors of thermitase while the chloromethyl ketone derivatives in general inhibit the enzyme irreversibly. The affinity increases by elongation of the peptide chain up to 4 residues indicating an interaction of the ligand with an extended binding site in thermitase. The P1 residue is phenylalanine in most of the inhibitors, according to the substrate specificity of thermitase for phenylalanine and other hydrophobic amino acids. Peptide ketones protected with the hydrophobic bencyloxycarbonyl group at the N-terminus are bound to the enzyme more tightly than the corresponding acetyl compounds. As reversible inhibitors peptide methyl ketones form a tetrahedral hemiketale with the oxygen of the active serine. In the case of the chloromethyl ketones this complex is stabilized by covalent binding to histidine-64. The inductive effect of the halogen favors the formation of the hemiketale and is expressed in .apprx. 20 times lower inhibition constants of the chloromethyl ketones. Kinetic experiments show a corresponding behavior in effector binding by thermitase and subtilisin in the S region, and from this point of view also indicate a high similarity of the active centers of both enzymes.

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