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The cobra venom factor-dependent C3 convertase of human complement. A kinetic and thermodynamic analysis of a protease acting on its natural high molecular weight substrate.

C. Vogel,H. Müller-Eberhard

Published 1982 in Journal of Biological Chemistry

ABSTRACT

Enzymes of the complement system exhibit, unlike most other proteases, a high substrate specificity, hy- drolyzing only a single peptide bond in their protein substrates. This property allowed the performance of a detailed analysis of the enzymatic activity of a protease acting on its natural high molecular weight sub- strate. The enzyme investigated was the cobra venom factor-dependent C3 convertase (EC 3.4.21.47) of hu- man complement. The enzyme constitutes a bimolecular complex of cobra venom factor and the catalytic site bearing fragment Bb of human Factor B. It hydrolyzes peptide bond 77 (Arg-Ser) of the a-chain of the human complement protein C3, thereby producing the fragments C3a and C3b. The enzyme was generated from isolated proteins. It exhibited spontaneous decay-dissociation into its subunits with a half-life of 7 h at 37 "C. The following kinetic parameters for C3 hydrolysis were determined: the Michaelis constant, K,, the catalytic constant, kc,,, the turnover number, the catalytic cycle time, the specific activity, the apparent second order rate constant, k,,,/K,, and the apparent first order rate constant for the low substrate concentration range. The encounter of enzyme and substrate pro-ceeded under rapid equilibrium conditions. For the for- mation of the enzyme-substrate complex, the equilibrium constant, K, the standard enthalpy, AH", standard entropy, AS", and standard Gibbs energy, AG", were determined. For the rate-limiting step of the overall reaction, the activation energy, E,, activation enthalpy, AH*, activation entropy, AS*, and Gibbs energy of activation, AG*, were derived. The results demonstrate that action of a protease of high molecular weight (Mr = 210,000) on its substrate of high molecular weight (Mr = 185,000) can be described in terms of Michaelis-Menten kinetics. The data are consistent with a double intermediate catalytic mechanism and a kinetic mechanism of a Tetra Uni Ping Pang Bi Bi reaction reduced to a Uni Bi reaction and therefore support the serine protease concept of Factor B-derived enzymes.

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