Functional role of the noncatalytic subunit of complement C5 convertase.
The C5 convertase is a serine protease that consists of two subunits: a catalytic subunit which is bound in a Mg2+-dependent complex to a noncatalytic subunit. To understand the functional role of the noncatalytic subunit, we have determined the C5-cleaving properties of the cobra venom factor-dependent C5 convertase (CVF, Bb) made with CVF purified from the venom of Naja naja (CVFn) and Naja haje (CVFh) and compared them to those for two C3b-dependent C5 convertases (ZymC3b,Bb and C3b,Bb). A comparison of the kinetic parameters indicated that although the four C5 convertases (CVFn,Bb, ZymC3b,Bb, CVFh,Bb, and C3b,Bb) had similar catalytic rate constants (kcat = 0.004-0.012 s-1) they differed 700-fold in their affinity for the substrate as indicated by the Km values (CVFn,Bb = 0.036 microM, ZymC3b,Bb = 1.24 microM, CVFh,Bb = 14.0 microM, and C3b,Bb = 24 microM). Analysis of binding interactions between C5 and the noncatalytic subunits (CVFh or C3b, or CVFn) using the BIAcore, revealed dissociation binding constants (Kd) that were similar to the Km values of the respective enzymes. The kinetic and binding data demonstrate that the binding site for C5 resides in the noncatalytic subunit of the enzyme, the affinity for the substrate is solely determined by the noncatalytic subunit and the catalytic efficiency of the enzyme appears not to be influenced by the nature of this subunit.
Rawal
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Department of Biochemistry, University of Texas Health Science Center, Tyler, TX 75708, USA.
Pangburn
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MK
D000818:Animals; D001665:Binding Sites / Q000276:immunology; D015374:Biosensing Techniques; D020134:Catalytic Domain / Q000276:immunology*; D050577:Complement C3-C5 Convertases / Q000097:blood / Q000378:metabolism / Q000502:physiology*; D011415:Complement Factor B / Q000378:metabolism; D003170:Complement Pathway, Alternative / Q000276:immunology; D004306:Dose-Response Relationship, Immunologic; D004546:Elapid Venoms / Q000378:metabolism; D017815:Elapidae / Q000276:immunology; D006801:Humans; D007700:Kinetics; D013329:Structure-Activity Relationship
D004546:Elapid Venoms; C016310:cobra venom factor; D050577:Complement C3-C5 Convertases; D011415:Complement Factor B