Edmiston P L, Schavolt K L, Kersteen E A, Moore N R, Borders C L
Department of Chemistry, College of Wooster, 44691, Wooster, OH, USA.
Biochim Biophys Acta. 2001 Apr 7;1546(2):291-8. doi: 10.1016/s0167-4838(01)00159-5.
Sequence homology analysis reveals that arginine-95 is fully conserved in 29 creatine kinases sequenced to date, but fully conserved as a tyrosine residue in 16 arginine kinases. Site-directed mutants of rabbit muscle creatine kinase (rmCK) were prepared in which R95 was replaced by a tyrosine (R95Y), alanine (R95A), or lysine (R95K). Kinetic analysis of phosphocreatine formation for each purified mutant showed that recombinant native rmCK and all R95 mutants follow a random-order, rapid-equilibrium mechanism. However, we observed no evidence for synergism of substrate binding by the recombinant native enzyme, as reported previously [Maggio et al., (1977) J. Biol. Chem. 252, 1202-1207] for creatine kinase isolated directly from rabbit muscle. The catalytic efficiencies of R95Y and R95A are reduced approximately 3000- and 2000-fold, respectively, compared to native enzyme, but that of R95K is reduced only 30-fold. The major contribution to the reduction of the catalytic efficiency of R95K is a 5-fold reduction in the affinity for creatine. This suggests that while a basic residue is required at position 95 for optimal activity, R95 is not absolutely essential for binding or catalysis in CK. R95Y has a significantly lower affinity for creatine than the native enzyme, but it also displays a somewhat lower affinity for MgATP and 100-fold reduction in k(cat). Interestingly, R95A appears to bind either creatine or MgATP first with affinities similar to those for the native enzyme, but it has a 10-fold lower affinity for the second substrate, suggesting that replacement of R95 by an alanine disrupts the active site organization and reduces the efficiency of formation of the catalytically competent ternary complex.
序列同源性分析表明，在迄今测序的29种肌酸激酶中，精氨酸95完全保守，但在16种精氨酸激酶中则完全保守为酪氨酸残基。制备了兔肌肉肌酸激酶（rmCK）的定点突变体，其中R95被酪氨酸（R95Y）、丙氨酸（R95A）或赖氨酸（R95K）取代。对每个纯化突变体的磷酸肌酸形成进行动力学分析表明，重组天然rmCK和所有R95突变体均遵循随机顺序、快速平衡机制。然而，我们没有观察到重组天然酶底物结合协同作用的证据，正如之前[Maggio等人，（1977年）《生物化学杂志》252卷，1202 - 1207页]报道的直接从兔肌肉中分离的肌酸激酶那样。与天然酶相比，R95Y和R95A的催化效率分别降低了约3000倍和2000倍，但R95K仅降低了30倍。R95K催化效率降低的主要原因是对肌酸的亲和力降低了5倍。这表明虽然95位需要一个碱性残基以实现最佳活性，但R95对于CK中的结合或催化并非绝对必要。R95Y对肌酸的亲和力明显低于天然酶，但它对MgATP的亲和力也略低，且催化常数（k(cat)）降低了100倍。有趣的是，R95A似乎首先以与天然酶相似的亲和力结合肌酸或MgATP，但它对第二种底物的亲和力低10倍，这表明用丙氨酸取代R95会破坏活性位点的组织，并降低催化活性三元复合物形成的效率。
