Inhibitory specificity of the anti-inflammatory myxoma virus serpin, SERP-1.
SERP-1 is a myxoma virus-encoded serpin, secreted from infected cells, that is required for virulence and has anti-inflammatory activity. We report that purified recombinant SERP-1 forms SDS-stable complexes with urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasmin, thrombin, and factor Xa. N-terminal sequencing confirmed Arg319-Asn320 as the site of reaction. Mutation of these residues to Ala-Ala abolished inhibitory activity but had no effect on the specific cleavage at Thr315-Leu316 seen with elastase and with cathepsin G. Kinetic analysis of the reactions with uPA, tPA, plasmin, thrombin, Xa, and C1s showed second-order rate constants to vary over 3 logs, from kinh = 3 x 10(5) M-1 s-1 with thrombin to approximately 600 M-1 s-1 with C1s, while steady-state inhibition constants ranged from KI = 10 pM with thrombin to approximately 100 nM with C1s. Stoichiometries of inhibition varied between SI = 1.4 +/- 0.1 for uPA to SI = 13 +/- 3 for thrombin. Analysis of the variations in inhibition kinetics shows that when serpins act at low concentrations, comparable with the target protease or with KI (as appears likely for SERP-1 in vivo), inhibitory specificity becomes less dominated by kinh and is increasingly dependent on partitioning within the branched reaction mechanism and on the lifetime of the inhibited complex.
Nash
P
P
Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Whitty
A
A
Handwerker
J
J
Macen
J
J
McFadden
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D015951:Factor Xa / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D007249:Inflammation; D016297:Mutagenesis, Site-Directed; D009233:Myxoma virus / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015843:Serpins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D015843:Serpins; D014764:Viral Proteins; D013917:Thrombin; D015951:Factor Xa; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator