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 K D of ≥2.5 μM and that Factor D acts on this complex with a second...

Full description

Saved in:

Bibliographic Details
Published in:	Biochemistry (Easton) Vol. 38; no. 9; pp. 2849 - 2859
Main Authors:	Taylor, Frederick R, Bixler, Sarah A, Budman, Joe I, Wen, Dingyi, Karpusas, Michael, Ryan, Sarah T, Jaworski, Gary J, Safari-Fard, Abbas, Pollard, Stuart, Whitty, Adrian
Format:	Journal Article
Language:	English
Published:	United States American Chemical Society 02-03-1999
Subjects:	Acetylation Anilides - chemistry Arginine - analogs & derivatives Arginine - chemistry Binding Sites Biotinylation Chromogenic Compounds - chemistry Complement Factor B - antagonists & inhibitors Complement Factor B - chemistry Complement Factor D - chemistry Complement Factor D - metabolism Enzyme Activation Humans Hydrolysis Models, Molecular Oligopeptides - chemistry Serine Endopeptidases - chemistry Serine Endopeptidases - metabolism Substrate Specificity
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	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 K D of ≥2.5 μM and that Factor D acts on this complex with a second-order rate constant of k cat/K M ≥ 2 × 106 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 103−104-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 (k cat/K M ≤ 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.
Bibliography:	istex:ADC585C233B6CCC226A1EF640ED4459D16BA0AA5 ark:/67375/TPS-VZJZ30W9-K ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0006-2960 1520-4995
DOI:	10.1021/bi982140f