title: The C3 convertase of the alternative pathway of human complement. Enzymic properties of the bimolecular proteinase.
abstract: The association of Factor B with C3b (the major fragment of complement component C3) in the presence of Mg2+ results in the formation of a bimolecular zymogen, C3b,B, which is activated by the serine proteinase Factor D, generating the C3 convertase, C3b,Bb (EC 3.4.21.47). Cleavage of native C3 by the C3 convertase was monitored by recording the increase in fluorescence associated with C3b formation in the presence of the fluorescent probe 8-anilinonaphthalene-1-sulphonate. Measurements of initial rates of C3b formation at various C3 concentrations were analysed in accordance with the Michaelis-Menten equation, yielding kcat. = 1.78 +/- 0.08 s-1, Km = 5.86 X 10(-6) M and turnover number = 107 min-1. The assay was used to measure the Ki values of a variety of proteinase inhibitors. The C3 convertase has a short half-life, owing to spontaneous dissociation of the complex. The t1/2 and kcat./Km of the enzyme were determined by fitting an equation modelling both the kinetic reaction and enzyme decay to the fluorimetrically measured progress curve. The enzyme, C3b,Bb, exhibited a t1/2 of 90 +/- 2 s and a kcat./Km of 31.1 X 10(4) +/- 0.8 X 10(4) M-1 X s-1 at physiological pH, ionic strength and temperature. The enzyme that initiates activation of the alternative pathway, C3(H2O),Bb, was also examined. It was slightly less stable (t1/2 = 77 +/- 3 s) and exhibited only half the activity of C3b,Bb (kcat./Km = 16.3 X 10(4) +/- 1.0 X 10(4) M-1 X s-1).
mesh_terms: D003166:Complement Activating Enzymes / Q000378:metabolism*; D003167:Complement Activation; D050577:Complement C3-C5 Convertases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003179:Complement C3b / Q000378:metabolism; D011415:Complement Factor B / Q000096:biosynthesis; D003170:Complement Pathway, Alternative; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006207:Half-Life; D007700:Kinetics; D008954:Models, Biological; D013050:Spectrometry, Fluorescence
chemical_list: D006146:Guanidines; D003179:Complement C3b; D003166:Complement Activating Enzymes; D050577:Complement C3-C5 Convertases; D011415:Complement Factor B; D019791:Guanidine