Chimerism reveals a role for the streptokinase β-domain in nonproteolytic active site formation, substrate, and inhibitor interactions

被引:11
作者
Gladysheva, IP
Sazonova, IY
Chowdhry, SA
Liu, L
Turner, RB
Reed, GL
机构
[1] Harvard Univ, Sch Publ Hlth, Cardiovasc Biol Lab, Boston, MA 02114 USA
[2] Massachusetts Gen Hosp, Boston, MA 02115 USA
关键词
D O I
10.1074/jbc.M202999200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Streptokinase (SK) and staphylokinase form cofactorenzyme complexes that promote the degradation of fibrin thrombi by activating human plasminogen. The unique abilities of streptokinase to nonproteolytically activate plasminogen or to alter the interactions of plasmin with substrates and inhibitors may be the result of high affinity binding mediated by the streptokinase beta-domain. To examine this hypothesis, a chimeric streptokinase, SKbetaswap, was created by swapping the SK beta-domain with the homologous beta-domain of Streptococcus uberis Pg activator (SUPA or PauA, SK uberis), a streptokinase that cannot activate human plasminogen. SKbetaswap formed a tight complex with microplasminogen with an affinity comparable with streptokinase. The SKbetaswap-plasmin complex also activated human plasminogen with catalytic efficiencies (k(cat)/K-m = 16.8 versus 15.2 muM(-1) min(-1)) comparable with streptokinase. However, SKbetaswap was incapable of nonproteolytic active site generation and activated plasminogen by a staphylokinase mechanism. When compared with streptokinase complexes, SKbetaswap-plasmin and SKbetaswap-microplasmin complexes had altered affinities for low molecular weight substrates. The SKbetaswap-plasmin complex also was less resistant than the streptokinase-plasmin complex to inhibition by alpha(2)-antiplasmin and was readily inhibited by soybean trypsin inhibitor. Thus, in addition to mediating high affinity binding to plasmin(ogen), the streptokinase beta-domain is required for nonproteolytic active site generation and specifically modulates the interactions of the complex with substrates and inhibitors.
引用
收藏
页码:26846 / 26851
页数:6
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