RUI: Creatine Kinase: Structure/Function Relationships of Conserved Residues by Site-Directed Mutagenesis

9982401边界肌酸激酶(CK)是兴奋性组织中的一种关键酶，这些组织需要大量的能量流，即骨骼肌、心肌和脑。CK催化三磷酸腺苷对肌酸的可逆磷酸化，生成磷酸肌酸和二磷酸腺苷。CK在可兴奋组织中的主要作用是允许细胞适应快速产生或耗尽ATP的活动，而不会在[ATP]/[ADP]的狭窄范围内显著波动，这是细胞最佳功能所必需的。这项拟议的研究将有助于更好地了解这种重要酶的作用机制。这项工作还将有助于更广泛地了解酶/蛋白质化学的其他基本领域，包括底物结合、催化和伴随的过渡态稳定、亚单位-亚单位结合形成低聚蛋白质以及“埋藏”的精氨酸在蛋白质结构稳定中的作用等因素。CK是一个更大的磷酸原激活酶(PKs)家族的成员，它使用镁ATP可逆地磷酸化一种胍基底物。CK是脊椎动物中唯一发现的PK，而精氨酸激酶(AK)是无脊椎动物中发现的主要PK。对25个CKs和12个AKs的序列同源性分析表明，71个残基在所有的PKs中都是完全保守的，这表明这些残基在所有的鸟嘌呤基激酶中都发挥着类似的作用。另有19个残基在CK中完全保守，在AK中作为一个不同的残基完全保守，表明这些残基可能在区分底物特异性和四级结构方面发挥作用(AK是单体，而CK是同源二聚体)。这种序列同源性分析，以及对具有结合过渡态模拟络合物和两个不同CKK的AK的X射线晶体结构的检查，已被用来提出保守残基的特定作用。兔肌肉CK的定点突变将被用于检测涉及以下方面的残基：1)底物结合；2)催化；3)过渡态稳定；4)单体-单体接触，目标是形成催化活性的CK单体；5)稳定CK亚基的三级结构，最初的重点是两个埋藏的“结构性”精氨酸；和6)肌酸结合，目的是改变酶的胍基底物专一性。每个突变体的特征是对“正向”反应(磷酸肌酸的形成)和必要时的“反向”反应(肌酸的形成)进行广泛的动力学分析。圆二色谱和差示扫描量热法将用于检测每个突变体的热稳定性，而添加核苷酸对固有蛋白质荧光的猝灭将用于确定低活性突变体的核苷酸亲和力和降低的过渡态稳定性。最后，选定的突变体的X射线晶体结构将与弗雷德里克癌症研究所的J·K·莫哈纳·拉奥博士合作确定。

9982401BordersCreatine kinase (CK) is a key enzyme in excitable tissues, which require large energy fluxes, i.e., skeletal muscle, heart muscle, and brain. CK catalyzes the readily reversible phosphorylation of creatine by ATP to produce phosphocreatine and ADP. The major role of CK in excitable tissues is to allow the cells to accommodate rapid-ATP-producing or -ATP-depleting activities without significant fluctuation in the narrow [ATP]/[ADP] range that is necessary for optimal cell function. The proposed research would contribute to a better understanding of the mechanism of this important enzyme. The work would also contribute to a broader understanding of other fundamental areas of enzyme/protein chemistry, including the factors involved in substrate binding, catalysis and accompanying transition-state stabilization, subunit-subunit association to form oligomeric proteins, and the role of "buried" arginines in the stabilization of protein structure. CK is a member of a larger family of phosphagen kinases (PKs) that use MgATP to reversibly phosphorylate a guanidino substrate. CK is the only PK found in vertebrates, while arginine kinase (AK) is the major PK found in invertebrates. Sequence homology analysis of 25 CKs and 12 AKs has identified 71 residues that are fully conserved in all PKs, suggesting that these residues play a similar role in all guanidino kinases. Another 19 residues are fully conserved in CK and fully conserved as a different residue in AK, suggesting that these residues may play roles in differentiating substrate specificity and quaternary structure (AK is a monomer, while CK is a homodimer). This sequence homology analysis, along with the examination of the x-ray crystal structures of an AK with a bound transition-state analog complex and two different CKs, has been used to propose specific roles for the conserved residues.Site-directed mutagenesis of rabbit muscle CK will be used to examine residues involved in: 1) substrate binding; 2) catalysis; 3) transition-state stabilization; 4) monomer-monomer contacts, with the goal of forming a catalytically-active CK monomer; 5) stabilization of the tertiary structure of CK subunits, with the initial focus on two buried "structural" arginines; and 6) creatine binding, with the goal of altering the guanidino substrate specificity of the enzyme. Each mutant will be characterized by extensive kinetic analysis of the "forward" reaction (phosphocreatine formation) and, where desirable, the "reverse" reaction (creatine formation). Circular dichroic spectra and differential scanning calorimetry will be used to examine the thermal stability of each mutant, while quenching of intrinsic protein fluorescence by added nucleotide will be used to determine the nucleotide affinity and diminished transition-state stabilization of low-activity mutants. Finally, the x-ray crystal structures of selected mutants will be determined in collaboration with Dr. J. K. Mohana Rao at the Frederick Cancer Institute.