Londergan Casey H, Baskin Rachel, Bischak Connor G, Hoffman Kevin W, Snead David M, Reynoso Christopher
Department of Chemistry, Haverford College , 370 Lancaster Avenue, Haverford, Pennsylvania 19041, United States.
Biochemistry. 2015 Jan 13;54(1):83-95. doi: 10.1021/bi5008063. Epub 2014 Nov 6.
Symmetric and asymmetric crystal structures of the apo and transition state analogue forms, respectively, of the dimeric rabbit muscle creatine kinase have invoked an "induced fit" explanation for asymmetry between the two subunits and their active sites. However, previously reported thiol reactivity studies at the dual active-site cysteine 283 residues suggest a more latent asymmetry between the two subunits. The role of that highly conserved active-site cysteine has also not been clearly determined. In this work, the S-H vibrations of Cys283 were observed in the unmodified MM isoform enzyme via Raman scattering, and then one and both Cys283 residues in the same dimeric enzyme were modified to covalently attach a cyano group that reports on the active-site environment via its infrared CN stretching absorption band while maintaining the catalytic activity of the enzyme. Unmodified and Cys283-modified enzymes were investigated in the apo and transition state analogue forms of the enzyme. The narrow and invariant S-H vibrational bands report a homogeneous environment for the unmodified active-site cysteines, indicating that their thiols are hydrogen bonded to the same H-bond acceptor in the presence and absence of the substrate. The S-H peak persists at all physiologically relevant pH's, indicating that Cys283 is protonated at all pH's relevant to enzymatic activity. Molecular dynamics simulations identify the S-H hydrogen bond acceptor as a single, long-resident water molecule and suggest that the role of the conserved yet catalytically unnecessary thiol may be to dynamically rigidify that part of the active site through specific H-bonding to water. The asymmetric and broad CN stretching bands from the CN-modified Cys283 suggest an asymmetric structure in the apo form of the enzyme in which there is a dynamic exchange between spectral subpopulations associated with water-exposed and water-excluded probe environments. Molecular dynamics simulations indicate a homogeneous orientation of the SCN probe group in the active site and thus rule out a local conformational explanation at the residue level for the multipopulation CN stretching bands. The homogeneous simulated SCN orientation suggests strongly that a more global asymmetry between the two subunits is the cause of the CN probe's broad and asymmetric infrared line shape. Together, these spectral observations localized at the active-site cysteines indicate an intrinsic, dynamic asymmetry between the two subunits that exists already in the apo form of the dimeric creatine kinase enzyme, rather than being induced by the substrate. Biochemical and methodological consequences of these conclusions are considered.
二聚体兔肌肌酸激酶的脱辅基形式和过渡态类似物形式分别具有对称和不对称的晶体结构，这引发了对两个亚基及其活性位点之间不对称性的“诱导契合”解释。然而，先前报道的在双活性位点半胱氨酸283残基处的硫醇反应性研究表明，两个亚基之间存在更潜在的不对称性。那个高度保守的活性位点半胱氨酸的作用也尚未明确确定。在这项工作中，通过拉曼散射在未修饰的MM同工型酶中观察到Cys283的S-H振动，然后在同一二聚体酶中的一个和两个Cys283残基被修饰，以共价连接一个氰基基团，该基团通过其红外CN伸缩吸收带报告活性位点环境，同时保持酶的催化活性。对未修饰和Cys283修饰的酶进行了脱辅基形式和过渡态类似物形式的研究。狭窄且不变的S-H振动带表明未修饰的活性位点半胱氨酸具有均匀的环境，这表明在有和没有底物的情况下，它们的硫醇都与同一个氢键受体形成氢键。S-H峰在所有生理相关的pH值下都持续存在，表明Cys283在与酶活性相关的所有pH值下都被质子化。分子动力学模拟确定S-H氢键受体为单个长期存在的水分子，并表明保守但催化上不必要的硫醇的作用可能是通过与水的特定氢键作用使活性位点的该部分动态刚性化。来自CN修饰的Cys283的不对称且宽泛的CN伸缩带表明该酶的脱辅基形式具有不对称结构，其中与水暴露和水排除的探针环境相关的光谱亚群之间存在动态交换。分子动力学模拟表明SCN探针基团在活性位点具有均匀的取向，因此排除了在残基水平上对多群体CN伸缩带的局部构象解释。均匀的模拟SCN取向强烈表明，两个亚基之间更全局的不对称性是CN探针宽泛且不对称的红外线形的原因。总之，这些定位于活性位点半胱氨酸的光谱观察结果表明，二聚体肌酸激酶酶的脱辅基形式中两个亚基之间已经存在内在的动态不对称性，而不是由底物诱导产生的。本文考虑了这些结论的生化和方法学后果。
