A NOVEL SUBSTRATE-BINDING POCKET INTERACTION RESTRICTS THE SPECIFICITY OF THE HUMAN NK CELL-SPECIFIC SERINE-PROTEASE, MET-ASE-1

Human Met-ase-1 is a NK cell-specific member of a family of serine pro teases (granzymes) that participate in target cell death inflicted by cytotoxic lymphocytes, This granzyme is predicted to cleave to the car boxyl side of long narrow hydrophobic amino acids (such as methionine) , but not large, bulky hydrophobic amino acids (such as phenylalanine) . To study the key structural features that confer this unusual serine protease specificity, active recombinant human Met-ase-1 was expresse d in COS-7 cells, Protease assays of transfected COS-7 cell lysates pr ovided evidence that an activation prohexapeptide normally regulates p rocessing of this granzyme in NK cells, Recombinant human Met-ase-1 cl eaved thiobenzylester substrates specifically after methionine, norleu cine, or leucine residues in the primary substrate site (P-1), Two key residues of human Met-ase-1, Lys179 Met (similar to chymotrypsin CHA1 92) and Ser201Gly (similar to CHA216), were mutated based upon a model structure derived from the crystal structure of chymotrypsin A. These mutants had reduced activity for substrate containing methionine at P -1, but acquired chymase activity for phenylalanine at P-1. Lys179 Met and Ser201Gly in the substrate-binding pocket of human Met-ase-1 rest rict the preference of this granzyme for long narrow hydrophobic amino acids in the P-1. A potential hydrogen-bonding interaction between th ese two residues on opposing sides of the substrate-binding pocket rep resents a novel molecular mechanism by which lymphocyte serine proteas es might provide greater substrate specificity.