Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins.
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).
Odake
S
S
School of Chemistry, Georgia Institute of Technology, Atlanta 30332.
Kam
C M
CM
Narasimhan
L
L
Poe
M
M
Blake
J T
JT
Krahenbuhl
O
O
Tschopp
J
J
Powers
J C
JC
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
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