Jourden Michael J, Geiss Paul R, Thomenius Michael J, Horst Lindsay A, Barty Melissa M, Brym Melissa J, Mulligan Guy B, Almeida Ryan M, Kersteen Betsy A, Myers Nichole R, Snider Mark J, Borders Charles L, Edmiston Paul L
Department of Chemistry, College of Wooster, Wooster, OH 44691, USA.
Biochim Biophys Acta. 2005 Aug 10;1751(2):178-83. doi: 10.1016/j.bbapap.2005.06.002.
Six fully conserved arginine residues (R129, R131, R235, R291, R319, and R340) closely grouped in the nucleotide binding site of rabbit muscle creatine kinase (rmCK) were mutated; four to alanine and all six to lysine. Kinetic analyses in the direction of phosphocreatine formation showed that all four alanine mutants led to substantial losses of activity with three (R129A, R131A, and R235A) having no detectable activity. All six lysine mutants retained variable degrees of reduced enzymatic activity. Static quenching of intrinsic tryptophan fluorescence was used to measure the binding constants for MgADP and MgATP. Nucleotide binding was at most only modestly affected by mutation of the arginine residues. Thus, the cluster of arginines seem to be primarily responsible for transition state stabilization which is further supported by the observation that none of the inactive mutants demonstrated the ability to form a transition analogue complex of MgADP.nitrate.creatine as determined by fluorescence quenching assays. As a whole, the results suggest that the most important role these residues play is to properly align the substrates for stabilization of the phosphoryl transfer reaction.
兔肌肉肌酸激酶（rmCK）核苷酸结合位点中紧密聚集的六个完全保守的精氨酸残基（R129、R131、R235、R291、R319和R340）发生了突变；四个突变为丙氨酸，六个全部突变为赖氨酸。在磷酸肌酸形成方向上的动力学分析表明，所有四个丙氨酸突变体均导致活性大幅丧失，其中三个（R129A、R131A和R235A）没有可检测到的活性。所有六个赖氨酸突变体均保留了不同程度的酶活性降低。利用内在色氨酸荧光的静态猝灭来测量MgADP和MgATP的结合常数。精氨酸残基的突变对核苷酸结合的影响至多只是适度的。因此，精氨酸簇似乎主要负责过渡态稳定，荧光猝灭分析表明，无活性的突变体均未表现出形成MgADP·硝酸盐·肌酸过渡类似物复合物的能力，这进一步支持了上述观点。总体而言，结果表明这些残基发挥的最重要作用是使底物正确排列，以稳定磷酰基转移反应。
