Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins
Details
Serval ID
serval:BIB_AA9FE02320B0
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins
Journal
Biochemistry
ISSN
0006-2960 (Print)
Publication state
Published
Issued date
02/1991
Volume
30
Number
8
Pages
2217-27
Notes
Comparative Study
Journal Article
Research Support, U.S. Gov't, P.H.S. --- Old month value: Feb 26
Journal Article
Research Support, U.S. Gov't, P.H.S. --- Old month value: Feb 26
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).
Keywords
Amino Acid Sequence
Animals
Coumarins/*pharmacology
Cytotoxicity, Immunologic/*drug effects
Esters
Granzymes
Humans
Kinetics
Mice
Molecular Sequence Data
Oligopeptides/chemical synthesis/*pharmacology
Protease Inhibitors/pharmacology
Serine Endopeptidases/*metabolism
Serine Proteinase Inhibitors
Substrate Specificity
T-Lymphocytes, Cytotoxic/drug effects/*enzymology/immunology
Pubmed
Web of science
Create date
24/01/2008 16:18
Last modification date
20/08/2019 16:14