title: Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins.
abstract: The active site structures of human Q31 granzyme A, murine granzymes (A, B, C, D, E, and F), and human granzymes (A, B, and 3) isolated from cytotoxic T lymphocytes (CTL) were studied with peptide thioester substrates, peptide chloromethyl ketone, and isocoumarin inhibitors. Human Q31, murine, and human granzyme A hydrolyzed Arg- or Lys-containing thioesters very efficiently with kcat/KM of 10(4)-10(5) M-1 s-1. Murine granzyme B was found to have Asp-ase activity and hydrolyzed Boc-Ala-Ala-Asp-SBzl with a kcat/KM value of 2.3 X 10(5) M-1 s-1. The rate was accelerated 1.4-fold when the 0.05 M NaCl in the assay was replaced with CaCl2. The preparation of granzyme B also had significant activity toward Boc-Ala-Ala-AA-SBzl substrates, where AA was Asn, Met, or Ser [kcat/KM = (4-5) X 10(4) M-1 s-1]. Murine granzymes C, D, and E did not hydrolyze any thioester substrate but contained minor contaminating activity toward Arg- or Lys-containing thioesters. Murine granzyme F had small activity toward Suc-Phe-Leu-Phe-SBzl, along with some contaminating trypsin-like activity. Human Q31 granzyme A, murine, and human granzyme A were inhibited quite efficiently by mechanism-based isocoumarin inhibitors substituted with basic groups (guanidino or isothiureidopropoxy). Although the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inactivated these tryptases poorly, it was the best isocoumarin inhibitor for murine granzyme B (kobs/[I] = 3700-4200 M-1 s-1). Murine and human granzyme B were also inhibited by Boc-Ala-Ala-Asp-CH2Cl; however, the inhibition was less potent than that with DCI. DCI, 3-(3-amino-propoxy)-4-chloroisocoumarin, 4-chloro-3-(3-isothiureidopropoxy)isocoumarin, and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin inhibited Q31 cytotoxic T lymphocyte mediated lysis of human JY lymphoblasts (ED50 = 0.5-5.0 microM).
authors.affiliation: School of Chemistry, Georgia Institute of Technology, Atlanta 30332.
authors.forename: S
authors.initials: S
authors.lastname: Odake
authors.forename: C M
authors.initials: CM
authors.lastname: Kam
authors.forename: L
authors.initials: L
authors.lastname: Narasimhan
authors.forename: M
authors.initials: M
authors.lastname: Poe
authors.forename: J T
authors.initials: JT
authors.lastname: Blake
authors.forename: O
authors.initials: O
authors.lastname: Krahenbuhl
authors.forename: J
authors.initials: J
authors.lastname: Tschopp
authors.forename: J C
authors.initials: JC
authors.lastname: Powers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003374:Coumarins / Q000494:pharmacology*; D003602:Cytotoxicity, Immunologic / Q000187:drug effects*; D004952:Esters; D053804:Granzymes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors; D013379:Substrate Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology
chemical_list: D003374:Coumarins; D004952:Esters; D009842:Oligopeptides; D011480:Protease Inhibitors; D015842:Serine Proteinase Inhibitors; D053804:Granzymes; D012697:Serine Endopeptidases; C000597315:granzyme A, mouse; C509212:GZMA protein, human