CREATINE-KINASE - THE REACTIVE CYSTEINE IS REQUIRED FOR SYNERGISM BUT IS NONESSENTIAL FOR CATALYSIS
CREATINE-KINASE - THE REACTIVE CYSTEINE IS REQUIRED FOR SYNERGISM BUT IS NONESSENTIAL FOR CATALYSIS
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DOI：
10.1021/bi00078a030
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发表时间：
1993-07-13
期刊：
影响因子：
2.9
通讯作者：
WALLIMANN, T
中科院分区：
文献类型：
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作者：
FURTER, R;FURTERGRAVES, EM;WALLIMANN, T
用巯基特异性试剂对兔肌肌酸激酶（CK）进行化学修饰，导致酶部分或完全失活。利用定点突变技术，我们将鸡心肌线粒体肌酸激酶（Mi（B）-CK）中相应的活性Cys 278分别用甘氨酸、丝氨酸、丙氨酸、天冬酰胺或天冬氨酸取代，得到的突变体Mi（B）-CK酶在其酶学性质上表现出类似的定性变化。在CK反应的两个方向上，观察到最佳pH值向较低值的偏移。突变型Mi（B）-CK在逆反应（ATP合成）中对游离ADP的抑制和在正反应（磷酸肌酸合成）中对游离ATP的抑制的敏感性增加几倍。除C278 D外，所有突变酶都能被氯离子和溴离子特异性激活。在相同条件下，C278 D和野生型Mi（B）-CK被显著抑制。在低氯离子浓度下，C278 D的V（max）比C278 N高约12倍。因此，Cys 278可能提供负电荷，其直接或间接参与使CK活性最大化。在接近最佳的逆反应条件下，与野生型Mi（B）-CK相比，突变体C278 G和C278 S的K（m）（PCr）增加了约11倍，但V（max）仅分别降低了1.7倍和2.8倍。因此，反应性半胱氨酸显然不是催化所必需的。对于兔肌肉CK，底物结合已显示为协同的（即，K（d）> K（m））。我们通过测定正向反应中两种底物的K（d）和K（m）值证实了野生型Mi（B）-CK的这一发现。对两种突变酶C278 G和C278 S的这些常数的分析表明，反应性半胱氨酸（1）不直接参与结合任一底物（突变体的K（d）值与野生型相比没有显著变化），（2）是协同底物结合所必需的（突变体的K（d）值小于相应的K（m）值）。这些结果表明，反应性半胱氨酸是必要的，赋予构象变化后，基板结合和支持的建议，该残基有一个作用，在塑造的活性位点，可能通过充当铰链之间的两个基板结合位点。
Chemical modification of rabbit muscle creatine kinase (CK) with thiol-specific reagents led to partial or complete inactivation of the enzyme. Using site-directed mutagenesis, we have substituted the corresponding reactive Cys278 in the chicken cardiac mitochondrial creatine kinase (Mi(b)-CK) with either glycine, serine, alanine, asparagine, or aspartate, The resulting mutant Mi(b)-CK enzymes showed qualitatively similar changes in their enzymatic properties. In both directions of the CK reaction, a shift of the pH optimum to lower values was observed. Mutant Mi(b)-CKs were severalfold more sensitive to inhibition by free ADP in the reverse reaction (ATP synthesis) and to free ATP in the forward reaction (phosphocreatine synthesis). With the exception of C278D, all mutant enzymes were specifically activated by chloride and bromide anions. C278D and wild-type Mi(b)-CK were significantly inhibited under the same conditions. At low chloride concentrations, the V(max) of C278D was about 12-fold higher than that of C278N. Thus, Cys278 probably provides a negative charge which is directly or indirectly involved in maximizing CK activity. Under near-optimal conditions in the reverse reaction, mutants C278G and C278S showed about an 11-fold increase in K(m)(PCr), but only 1.7- and 2.8-fold reductions in V(max), respectively, compared to wild-type Mi(b)-CK. Thus, the reactive cysteine clearly is not essential for catalysis. For rabbit muscle CK, substrate binding had been shown to be synergistic (i.e., K(d) > K(m)). We confirmed this finding for wild-type Mi(b)-CK by determining the K(d) and K(m) values for both substrates in the forward reaction. Analysis of these constants for the two mutant enzymes C278G and C278S showed that the reactive cysteine (1) is not directly involved in binding either substrate (K(d) values for mutants were not dramatically changed compared to the wild type) and (2) is necessary for synergistic substrate binding (K(d) values for mutants were smaller than the corresponding K(m) values). These results suggest that the reactive cysteine is necessary to confer conformational changes upon substrate binding and support the proposal that this residue has a role in shaping the active site, possibly by acting as a hinge between the two substrate binding sites.