CATALYTIC ROLE OF A SURFACE LOOP OF THE COMPLEMENT SERINE-PROTEASE FACTOR-D

We have investigated the structural determinants of the unique functio nal properties of complement factor D by constructing and testing a se ries of trypsin-like mutants of the enzyme. Mutational replacement of the primary substrate-binding pocket of factor D with that of trypsin resulted in a mutant (M1) with greatly reduced proteolytic activity an d slightly reduced reactivity toward small thioester substrates. Combi ning the M1 mutations with substitution of Tyr for Ser(94), previously shown to enhance substantially both the proteolytic and esterolytic a ctivities of factor D, produced a mutant (M2) with reactivities simila r to M1. Replacement of the surface loop formed by residues 184-188 of M1 and M2 with the corresponding loop of trypsin produced mutants exh ibiting one and two orders of magnitude higher esterolytic activity, r espectively, than native factor D. However, the proteolytic activity o f both mutants was similar to that of M1 and M2. We conclude that loop (184-188) is an important determinant of the geometry of the primary s pecificity pocket of factor D. The low proteolytic activity of these m utants supports the proposal that the proteolytically active conformat ion of factor D is induced by its natural substrate, C3bB.