C5 convertase of the alternative pathway of complement. Kinetic analysis of the free and surface-bound forms of the enzyme.
Although proteolytic activation of the complement protein C5 initiates important defensive and occasionally pathological inflammatory reactions, the enzymatic properties of the enzymes responsible for this cleavage have never been examined. We have studied the kinetic parameters of the C5 convertase of the alternative pathway of complement, either bound to a zymosan surface or in its monomeric soluble form. C5 convertase enzymatic activity was measured as a function of C5 concentration by quantitating production of C5b,6 under physiological conditions of temperature, pH, and ionic strength. The C5 convertases appeared to follow Michaelis-Menten kinetics and exhibited similar catalytic rate constants (kcat). However, the surface-bound enzyme, ZymC3b,Bb had a Km (1.4 microM) that was 17 times lower than that of the soluble monomeric form of the enzyme, C3b,Bb (Km = 24 microM). The kcat for the cell-bound enzyme, ZymC3b,Bb was 0.0048 s-1 and that for soluble C3b,Bb was 0.0110 s-1. Both forms of the enzyme had a low turnover number at Vmax (0.23 to 0.68 C5/min/enzyme). Substituting Mg2+ for Ni2+ did not alter the kinetic parameters but lowered the half-life of the enzyme by 5-7-fold. The kinetic data presented demonstrate that the fluid phase C5 convertase, C3b,Bb, can cleave C5 without the aid of a second C3b molecule. The results also show that the greater enzymatic activity previously observed for the surface-bound C5 convertases is not due to higher catalytic efficiency but is solely due to higher affinity for the substrate C5. In blood, C5 concentrations are 3-4-fold below the Km determined for the surface-bound C5 convertase suggesting a direct correlation between the local C5 concentration and production of the anaphylatoxin C5a and the cytolytic C5b-9 complex.
Rawal
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Department of Biochemistry, University of Texas Health Science Center, Tyler, Texas 75710, USA.
Pangburn
M K
MK
D002462:Cell Membrane / Q000201:enzymology; D003176:Complement C3 / Q000378:metabolism; D050577:Complement C3-C5 Convertases / Q000378:metabolism*; D003182:Complement C5 / Q000378:metabolism; D017242:Complement Factor H / Q000378:metabolism; D003170:Complement Pathway, Alternative; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009994:Osmolar Concentration; D013696:Temperature
C493495:CFH protein, human; D003176:Complement C3; D003182:Complement C5; D017242:Complement Factor H; D050577:Complement C3-C5 Convertases