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C1 Inhibitor: Analysis of the Role of Amino Acid Residues Within the Reactive Center Loop in Target Protease Recognition¹

Rana Zahedi^2,*, Ryan C. MacFarlane^*, Jeffrey J. Wisnieski and Alvin E. Davis, III^3,*

^* Center for Blood Research, Boston, MA 02115; and Department of Veterans Affairs Medical Center, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106

Previous analysis of a naturally occurring C1 inhibitor P2 mutant (Ala⁴⁴³Val) 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 (Ala⁴³⁹) and P8'/9' (Gln⁴⁵²Gln⁴⁵³). Ala⁴³⁹Val is a naturally occurring mutant observed in a patient with hereditary angioedema. Previous data suggested that Gln⁴⁵²Gln⁴⁵³ might be a contact site for C1s. Reactivity of the inhibitors toward target (C1s, C1r, kallikrein, factor XIIa, and plasmin) and nontarget proteases (-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 10⁵ M^-1s^-1 for the wild type to 1.61, 1.29, and 0.63 x 10⁵ for the Ser, Thr, and Val mutants, respectively. In contrast, the Ala⁴³⁹Val and the Gln⁴⁵²Gln⁴⁵³Ala 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 Gln⁴⁵²Gln⁴⁵³ comprise a portion of a protease contact site within the inhibitor.

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