Azzi Arezki, Clark Shawn A, Ellington W Ross, Chapman Michael S
Institute of Molecular Biophysics, Kasha Laboratory, Florida State University, Tallahassee, FL 32306-4380, USA.
Protein Sci. 2004 Mar;13(3):575-85. doi: 10.1110/ps.03428304.
Phosphagen kinases catalyze the reversible transfer of a phosphate between ATP and guanidino substrates, a reaction that is central to cellular energy homeostasis. Members of this conserved family include creatine and arginine kinases and have similar reaction mechanisms, but they have distinct specificities for different guanidino substrates. There has not been a full structural rationalization of specificity, but two loops have been implicated repeatedly. A small domain loop is of length that complements the size of the guanidino substrate, and is located where it could mediate a lock-and-key mechanism. The second loop contacts the substrate with a valine in the methyl-substituted guanidinium of creatine, and with a glutamate in the unsubstituted arginine substrate, leading to the proposal of a discriminating hydrophobic/hydrophilic minipocket. In the present work, chimeric mutants were constructed with creatine kinase loop elements inserted into arginine kinase. Contrary to the prior rationalizations of specificity, most had measurable arginine kinase activity but no creatine kinase activity or enhanced phosphocreatine binding. Guided by structure, additional mutations were introduced in each loop, recovering arginine kinase activities as high as 15% and 64% of wild type, respectively, even though little activity would be expected in the constructs if the implicated sites had dominant roles in specificity. An atomic structure of the mismatched complex of arginine kinase with creatine and ADP indicates that specificity can also be mediated by an active site that allows substrate prealignment that is optimal for reactivity only with cognate substrates and not with close homologs that bind but do not react.
磷酸原激酶催化ATP和胍基底物之间磷酸的可逆转移，该反应是细胞能量稳态的核心。这个保守家族的成员包括肌酸激酶和精氨酸激酶，它们具有相似的反应机制，但对不同胍基底物具有不同的特异性。目前尚未对特异性进行完整的结构解释，但有两个环被反复提及。一个小结构域环的长度与胍基底物的大小互补，且位于可以介导锁钥机制的位置。第二个环与肌酸甲基取代胍基中的缬氨酸以及未取代精氨酸底物中的谷氨酸接触，由此提出了一个区分性的疏水/亲水微口袋。在本研究中，构建了将肌酸激酶环元件插入精氨酸激酶的嵌合突变体。与之前对特异性的解释相反，大多数突变体具有可测量的精氨酸激酶活性，但没有肌酸激酶活性或增强的磷酸肌酸结合能力。在结构的指导下，在每个环中引入了额外的突变，分别恢复了高达野生型15%和64%的精氨酸激酶活性，尽管如果所涉及的位点在特异性中起主导作用，预计这些构建体中几乎没有活性。精氨酸激酶与肌酸和ADP不匹配复合物的原子结构表明，特异性也可以由一个活性位点介导，该活性位点允许底物预排列，这种预排列仅对同源底物具有最佳反应性，而对结合但不反应的密切同源物则不然。
