Varfaj Fatbardha, Wakabayashi Hironao, Fay Philip J
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, NY 14642, USA.
J Biol Chem. 2007 Jul 13;282(28):20264-72. doi: 10.1074/jbc.M701327200. Epub 2007 May 22.
Activated Protein C (APC) inactivates factor VIIIa by cleavage at Arg(336) and Arg(562) within the A1 and A2 subunits, respectively, with reaction at the former site occurring at a rate approximately 25-fold faster than the latter. Recombinant factor VIII variants possessing mutations within the P4-P3' sequences were used to determine the contributions of these residues to the disparate cleavage rates at the two P1 sites. Specific activity values for 336(P4-P3')562, 336(P4-P2)562, and 336(P1'-P3')562 mutants, where indicated residues surrounding the Arg(336) site were replaced with those surrounding Arg(562), were similar to wild type (WT) factor VIII; whereas 562(P4-P3')336 and 562(P4-P2)336 mutants showed specific activity values <1% the WT value. Inactivation rates for the 336 site mutants were reduced approximately 6-11-fold compared with WT factor VIIIa, and approached values attributed to cleavage at Arg(562). Cleavage rates at Arg(336) were reduced approximately 100-fold for 336(P4-P3')562, and approximately 9-16-fold for 336(P4-P2)562 and 336(P1'-P3')562 mutants. Inhibition kinetics revealed similar affinities of APC for WT factor VIIIa and 336(P4-P3')562 variant. Alternatively, the 562(P4-P3')336 variant showed a modest increase in cleavage rate ( approximately 4-fold) at Arg(562) compared with WT, whereas these rates were increased by approximately 27- and 6-fold for 562(P4-P3')336 and 562(P4-P2)336, respectively, using the factor VIII procofactor form as substrate. Thus the P4-P3' residues surrounding Arg(336) and Arg(562) make significant contributions to proteolysis rates at each site, apparently independent of binding affinity. Efficient cleavage at Arg(336) by APC is attributed to favorable P4-P3' residues at this site, whereas cleavage at Arg(562) can be accelerated following replacement with more optimal P4-P3' residues.
活化蛋白C（APC）通过分别切割A1和A2亚基内的精氨酸（336）和精氨酸（562）来使因子VIIIa失活，在前一个位点的反应速率比后一个位点快约25倍。利用在P4 - P3'序列内具有突变的重组因子VIII变体来确定这些残基对两个P1位点不同切割速率的贡献。336（P4 - P3'）562、336（P4 - P2）562和336（P1' - P3'）562突变体的比活性值，其中围绕精氨酸（336）位点的指示残基被围绕精氨酸（562）的残基取代，与野生型（WT）因子VIII相似；而562（P4 - P3'）336和562（P4 - P2）336突变体的比活性值<WT值的1%。与WT因子VIIIa相比，336位点突变体的失活速率降低了约6 - 11倍，并接近归因于在精氨酸（562）处切割的值。对于336（P4 - P3'）562，精氨酸（336）处的切割速率降低了约100倍，对于336（P4 - P2）562和336（P1' - P3'）562突变体降低了约9 - 16倍。抑制动力学显示APC对WT因子VIIIa和336（P4 - P3'）562变体具有相似的亲和力。另外，与WT相比，562（P4 - P3'）336变体在精氨酸（562）处的切割速率适度增加（约4倍），而使用因子VIII辅因子形式作为底物时，562（P4 - P3'）336和562（P4 - P2）336的这些速率分别增加了约27倍和6倍。因此，围绕精氨酸（336）和精氨酸（562）的P4 - P3'残基对每个位点的蛋白水解速率有显著贡献，显然与结合亲和力无关。APC在精氨酸（336）处的有效切割归因于该位点有利的P4 - P3'残基，而在用更优化的P4 - P3'残基取代后，精氨酸（562）处的切割可以加速。
