| Abstract
| - The role of basic residues of the 70−80-loop, Arg74, Arg75, and Lys78 (chymotrypsin numbering)in the catalytic function of activated protein C (APC) was investigated by expressing mutants of proteinC in which these residues were replaced with Ala in three separate constructs. Following purification tohomogeneity and activation by thrombin, the catalytic properties of the mutants were characterized withrespect to their ability to cleave the chromogenic substrate Spectrozyme PCa, react with protein C inhibitor(PCI), and inactivate factor Va. Relative to wild-type APC, the mutants cleaved Spectrozyme PCa withidentical or improved catalytic efficiencies. Similarly, PCI inhibited mutants with identical or improvedsecond-order rate constants (k2) in the absence of heparin. However, the heparin-catalyzed inhibition ofmutants by PCI was impaired ∼10-fold. Analysis of k2 values by a ternary complex model revealed thatthe affinities of mutants for heparin were impaired to a similar extent. Moreover, analysis of the NaClgradient elution profiles of APC derivatives from Heparin-Sepharose supported this conclusion. Anoligosaccharide containing 14 residues efficiently catalyzed the PCI inhibition of APC by a templatemechanism. Further studies revealed that the ability of Arg74 and Arg75 mutants to inactivate factor Vawas markedly impaired. We conclude that basic residues of the 70−80-loop are critical for the catalyticfunction of APC.
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