Characterization of Lys‐698 to met substitution in human plasminogen catalytic domain

Streptokinase (SK) is a human plasminogen (Pg) activator secreted by streptococci. The activation mechanism of SK differs from that of physiological Pg activators in that SK is not a protease and cannot proteolytically activate Pg. Instead, it forms a tight complex with Pg that proteolytically activates other Pg molecules. The residue Lys‐698 of human Pg was hypothesized to participate in triggering activation in the SK–Pg complex. Here, we report a study of the Lys‐698 to Met substitution in the catalytic domain of Pg (μPg) containing the proteolytic activation‐resistant background (R561A). While it remains competent in forming a complex with SK, maintaining a comparable equilibration dissociation constant ( K D ), the recombinant protein shows a nearly 60‐fold reduction in amidolytic activity relative to its R561A background when mixed with native SK. A 2.3 Å crystal structure of this mutant μPg confirmed the correct folding of this recombinant protein. Combined with other biochemical data, these results support the premise that Lys‐698 of human Pg plays a functional role in the so‐called N‐terminal insertion activation mechanism by SK. Proteins 2004. © 2004 Wiley‐Liss, Inc.