Induced fit activation mechanism of the exceptionally specific serine protease, complement Factor D

We have investigated the mechanism by which the complement protease, Factor D, achieves its high specificity for the cleavage of Factor B in complex w ith C3(H2O). Kinetic experiments showed that Factor B and C3(H2O) associate with a K-D of greater than or equal to 2.5 mu M and that Factor D acts on this complex with a second-order rate constant of k(cat)/K-M greater than o r equal to 2 x 10(6) M-1 s(-1), close to the rate of a diffusion-controlled reaction for proteins of this size. In contrast, Factor D, which is a memb er of the trypsin family of serine proteases, was 10(3)-10(4)-fold less act ive than trypsin toward both thioester and p-nitroanilide substrates contai ning an arginine at Fl. Furthermore, peptides spanning the Factor B cleavag e site were not detectably cleaved by Factor D (k(cat)/K-M less than or equ al to 0.5 M-1 s(-1)). These results imply that contacts between Factor D an d the C3(H2O)B complex, outside the vicinity of the cleavage site in Factor B, generate greater than or equal to 9 kcal/mol of binding energy to stabi lize the transition state for reaction. In support of this, we demonstrate that chemical modification of Factor D at a single lysine residue that is d istant from the active site abolishes the activity of the enzyme toward Fac tor B while not affecting activity toward small synthetic substrates. We pr opose that Factor D may exemplify a special case of the induced fit mechani sm in which the requirement for conformational activation of the enzyme res ults in a substantial increase in substrate specificity.