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 (microPg) 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 A crystal structure of this mutant microPg 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.
Terzyan
Simon
S
Crystallography Research Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, Oklahoma 73104, USA.
Wakeham
Nancy
N
Zhai
Peng
P
Rodgers
Karla
K
Zhang
Xuejun C
XC
D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D002863:Chromogenic Compounds / Q000378:metabolism; D018360:Crystallography, X-Ray; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008239:Lysine / Q000737:chemistry; D046911:Macromolecular Substances; D008715:Methionine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010958:Plasminogen / Q000737:chemistry* / Q000378:metabolism; D018583:Pliability; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013300:Streptokinase / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance
D002863:Chromogenic Compounds; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D010958:Plasminogen; D008715:Methionine; D013300:Streptokinase; D008239:Lysine