Induced Fit Activation Mechanism of the Exceptionally Specific Serine Protease, Complement Factor D^†

Abstract

We have investigated the mechanism by which the complement protease, Factor D, achieves its high specificity for the cleavage of Factor B in complex with C3(H2O). Kinetic experiments showed that Factor B and C3(H2O) associate with a KD of >/=2.5 microM and that Factor D acts on this complex with a second-order rate constant of kcat/KM >/= 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 member of the trypsin family of serine proteases, was 10(3)-10(4)-fold less active than trypsin toward both thioester and p-nitroanilide substrates containing an arginine at P1. Furthermore, peptides spanning the Factor B cleavage site were not detectably cleaved by Factor D (kcat/KM </= 0.5 M-1 s-1). These results imply that contacts between Factor D and the C3(H2O)B complex, outside the vicinity of the cleavage site in Factor B, generate >/=9 kcal/mol of binding energy to stabilize the transition state for reaction. In support of this, we demonstrate that chemical modification of Factor D at a single lysine residue that is distant from the active site abolishes the activity of the enzyme toward Factor B while not affecting activity toward small synthetic substrates. We propose that Factor D may exemplify a special case of the induced fit mechanism in which the requirement for conformational activation of the enzyme results in a substantial increase in substrate specificity.

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

“…As a consequence, peptide substrates are not sufficient to reveal the full catalytic potential of the enzymes (35). Interestingly, while cleavage of tri-, tetra-, and pentapeptides have been reported to be slow, the dipeptidase activity of these two enzymes is relatively efficient (35,37,39). For both factor D and C1r, we observed efficient cleavage of substrates with P 3 ϭ Arg or Lys (Fig.…”

Access to the Complement Factor B Scissile Bond Is Facilitated by Association of Factor B with C3b Protein

“…Although free factor B is not cleaved by factor D, it can be cleaved by other trypsin-like serine proteases such as plasmin (16). The double latch structure formed by the Arg 234 salt bridges serves to minimize such unproductive activity.…”

“…1, B and C) (15). The salt bridges sequester the scissile bond (between Arg 234 and Lys 235 ) and prevent unproductive cleavage of factor B by circulating serine proteases (16). It is not known how the double latch structure is opened to permit factor D cleavage of the scissile bond in the short lived C3bB(Mg 2ϩ ) complex.…”

Liver gene expression associated with diet and lesion development in atherosclerosis-prone mice: induction of components of alternative complement pathway

“…Although once thought to be expressed exclusively in adipocytes, adipsin mRNA is found in the gastrointestinal tract, spleen, and liver (16,29). Factor D is unique among complement components in that it does not require proteolytic processing for activation; and it exhibits exceptional specificity for its substrate C3bB (43). Because it also circulates in plasma at rate-limiting concentrations, serum factor D activity is a linear function of its transcription level (43).…”

“…Factor D is unique among complement components in that it does not require proteolytic processing for activation; and it exhibits exceptional specificity for its substrate C3bB (43). Because it also circulates in plasma at rate-limiting concentrations, serum factor D activity is a linear function of its transcription level (43). Thus our observation of highly induced factor D expression with high-fat feeding suggests a mechanism for increased peripheral activity of the alternative complement pathway, i.e., the C3b:B:E-LDL complex assembled in the vascular wall is activated by enhanced circulating concentrations of factor D. An independent study, also recently observing dramatic induc- The 102 genes with expression levels altered by diet [Pr(F) Յ 0.0005] were classified by primary biochemical function.…”