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Mutational analysis of the substrate binding site of human complement factor D
S Kim1, S V Narayana, J E Volanakis
1Department of Microbiology, University of Alabama at Birmingham 35294.
Biochemistry
|December 6, 1994
Summary
Researchers explored unique amino acid substitutions in complement factor D, a serine protease. Key residues were identified that influence substrate binding and catalytic activity, offering insights into complement system function.
Area of Science:
- Biochemistry
- Immunology
- Structural Biology
Background:
- Complement factor D is a serine protease with limited known substrates and low esterolytic activity.
- Its unique structure, revealed by X-ray crystallography, differs from other serine proteases in catalytic triad and substrate-binding regions.
Purpose of the Study:
- To investigate the role of specific amino acid substitutions in factor D's unique structural and functional characteristics.
- To identify residues critical for substrate binding and catalytic activity.
Main Methods:
- Constructed mutant factor D variants by substituting trypsin substrate-binding residues.
- Expressed wild-type and mutant factor D in Chinese hamster ovary cells.
- Purified recombinant proteins and performed hemolytic, proteolytic, and esterolytic assays.
Main Results:
- Mutant factor D proteins were successfully expressed and purified.
- Residues Thr-198, Ser-199, Arg-202, and potentially Val-203 were identified as determinants for substrate binding and catalysis.
- Experimental data support the hypothesis that substrate binding induces factor D's active conformation.
Conclusions:
- Specific amino acid residues in factor D are crucial for its substrate specificity and catalytic function.
- These findings enhance understanding of the complement system's enzymatic mechanisms and regulation.
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