title: Formation of high affinity C5 convertase of the classical pathway of complement.
abstract: C3/C5 convertase is a serine protease that cleaves C3 and C5. In the present study we examined the C5 cleaving properties of classical pathway C3/C5 convertase either bound to the surface of sheep erythrocytes or in its free soluble form. Kinetic parameters revealed that the soluble form of the enzyme (C4b,C2a) cleaved C5 at a catalytic rate similar to that of the surface-bound form (EAC1,C4b,C2a). However, both forms of the enzyme exhibited a poor affinity for the substrate, C5, as indicated by a high Km (6-9 microM). Increasing the density of C4b on the cell surface from 8,000 to 172,000 C4b/cell did not influence the Km. Very high affinity C5 convertases were generated only when the low affinity C3/C5 convertases (EAC1,C4b,C2a) were allowed to deposit C3b by cleaving native C3. These C3b-containing C3/C5 convertases exhibited Km (0.0051 microM) well below the normal concentration of C5 in blood (0.37 microM). The data suggest that C3/C5 convertase assembled with either monomeric C4b or C4b-C4b complexes are inefficient in capturing C5 but cleave C3 opsonizing the cell surface with C3b for phagocytosis. Deposition of C3b converts the enzymes to high affinity C5 convertases, which cleave C5 in blood at catalytic rates approaching Vmax, thereby switching from C3 to C5 cleavage. Comparison of the kinetic parameters with those of the alternative pathway convertase indicates that the 6-9-fold greater catalytic rate of the classical pathway C5 convertase may compensate for the fewer numbers of C5 convertase sites generated upon activation of this pathway.
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, Tyler, Texas 75703, USA. nenoo.rawal@uthct.edu
authors.forename: Nenoo
authors.initials: N
authors.lastname: Rawal
authors.forename: Michael K
authors.initials: MK
authors.lastname: Pangburn
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D003175:Complement C2 / Q000096:biosynthesis; D003176:Complement C3 / Q000096:biosynthesis; D050577:Complement C3-C5 Convertases / Q000737:chemistry*; D003181:Complement C4 / Q000096:biosynthesis; D003182:Complement C5 / Q000096:biosynthesis; D003183:Complement C6 / Q000096:biosynthesis; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012756:Sheep; D013696:Temperature; D013997:Time Factors
chemical_list: D003175:Complement C2; D003176:Complement C3; D003181:Complement C4; D003182:Complement C5; D003183:Complement C6; D003902:Detergents; D007477:Ions; D050577:Complement C3-C5 Convertases