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

RUI：肌酸激酶：通过定点诱变实现保守残基的结构/功能关系

• 批准号: 9982401
• 负责人: Charles Borders
• 金额: $ 19.23万
• 依托单位: College of Wooster
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9982401&HistoricalAwards=false
• 关键词: RUI; Creatine; Kinase; Structure; Function

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.

9982401 Borders肌酸激酶（CK）是可兴奋组织中的关键酶，其需要大的能量通量，即，骨骼肌、心肌和大脑。 CK催化肌酸被ATP可逆磷酸化产生磷酸肌酸和ADP。 CK在可兴奋组织中的主要作用是允许细胞适应快速产生ATP或消耗ATP的活动，而不会在最佳细胞功能所必需的狭窄[ATP]/[ADP]范围内发生显著波动。 这项研究将有助于更好地了解这种重要酶的作用机制。 这项工作也将有助于更广泛地了解酶/蛋白质化学的其他基本领域，包括参与底物结合，催化和伴随的过渡态稳定，亚基-亚基缔合形成寡聚蛋白质的因素，以及“埋藏”的蛋白质在稳定蛋白质结构中的作用。CK是磷酸原激酶（PK）的较大家族的成员，其使用MgATP可逆地磷酸化胍基底物。 CK是脊椎动物中发现的唯一PK，而精氨酸激酶（AK）是无脊椎动物中发现的主要PK。 25 CK和12 AK的序列同源性分析已经确定了71个残基，在所有的PK中是完全保守的，这表明这些残基在所有的胍基激酶中发挥类似的作用。 另外19个残基在CK中是完全保守的，在AK中作为不同的残基是完全保守的，这表明这些残基可能在区分底物特异性和四级结构（AK是单体，而CK是同源二聚体）方面起作用。 这种序列同源性分析，沿着与结合的过渡态类似物复合物和两种不同CK的AK的X-射线晶体结构的检查，已被用于提出保守残基的特定作用。4）单体-单体接触，目的是形成具有催化活性的CK单体; 5）CK亚基的三级结构的稳定化，最初关注于两个埋藏的“结构”酶;和6）肌酸结合，目的是改变酶的胍基底物特异性。 每种突变体的特征在于“正向”反应（磷酸肌酸形成）的广泛的动力学分析，并在需要时，“反向”反应（肌酸形成）。 圆二色性光谱和差示扫描量热法将用于检查每个突变体的热稳定性，而通过添加核苷酸的内在蛋白质荧光的淬灭将用于确定低活性突变体的核苷酸亲和力和减少的过渡态稳定性。 最后，选定的突变体的X射线晶体结构将与J. K。弗雷德里克癌症研究所的Mohana Rao。