C1 inhibitor: analysis of the role of amino acid residues within the reactive center loop in target protease recognition.
Previous analysis of a naturally occurring C1 inhibitor P2 mutant (Ala(443)-->Val) indicated a role for P2 in specificity determination. To define this role and that of other reactive center loop residues, a number of different amino acids were introduced at P2, as well as at P6 (Ala(439)) and P8'/9' (Gln(452)Gln(453)). Ala(439)-->Val is a naturally occurring mutant observed in a patient with hereditary angioedema. Previous data suggested that Gln(452)Gln(453) might be a contact site for C1s. Reactivity of the inhibitors toward target (C1s, C1r, kallikrein, beta factor XIIa, and plasmin) and nontarget proteases (alpha-thrombin and trypsin) were studied. Substitution of P2 with bulky or charged residues resulted in decreased reactivity with all target proteases. Substitution with residues with hydrophobic or polar side chains resulted in decreased reactivity with some proteases, but in unaltered or increased reactivity with others. Second order rate constants for the reaction with C1s were determined for the mutants with activities most similar to the wild-type protein. The three P2 mutants showed reductions in rate from 3.35 x 10(5) M(-1)s(-1) for the wild type to 1.61, 1.29, and 0.63 x 10(5) for the Ser, Thr, and Val mutants, respectively. In contrast, the Ala(439)-->Val and the Gln(452)Gln(453)-->Ala mutants showed little difference in association rates with C1s, in comparison with the wild-type inhibitor. The data confirm the importance of P2 in specificity determination. However, the P6 position appears to be of little, if any, importance. Furthermore, it appears unlikely that Gln(452)Gln(453) comprise a portion of a protease contact site within the inhibitor.
Zahedi
R
R
Center for Blood Research, 800 Huntington Avenue, Boston, MA 02115.
MacFarlane
R C
RC
Wisnieski
J J
JJ
Davis
A E
AE
D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D003174:Complement C1 Inactivator Proteins / Q000235:genetics / Q000378:metabolism*; D015923:Complement C1r / Q000378:metabolism; D003173:Complement C1s / Q000378:metabolism; D015956:Factor XIIa / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D007610:Kallikreins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D003174:Complement C1 Inactivator Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007610:Kallikreins; D012697:Serine Endopeptidases; D015956:Factor XIIa; D014357:Trypsin; D015923:Complement C1r; D003173:Complement C1s; D013917:Thrombin; D005341:Fibrinolysin