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 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.
Department of Protein Engineering, Biogen, Inc., Cambridge, Massachusetts 02142, USA.
F R
FR
Taylor
S A
SA
Bixler
J I
JI
Budman
D
D
Wen
M
M
Karpusas
S T
ST
Ryan
G J
GJ
Jaworski
A
A
Safari-Fard
S
S
Pollard
A
A
Whitty
D000107:Acetylation; D000813:Anilides / Q000737:chemistry; D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry; D001665:Binding Sites; D019921:Biotinylation; D002863:Chromogenic Compounds / Q000737:chemistry; D011415:Complement Factor B / Q000037:antagonists & inhibitors / Q000737:chemistry; D011416:Complement Factor D / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
D000813:Anilides; D002863:Chromogenic Compounds; D009842:Oligopeptides; D001120:Arginine; D012697:Serine Endopeptidases; C493488:CFD protein, human; D011416:Complement Factor D; D011415:Complement Factor B