The Molecular Mechanisms Governing the Catalysis of 6-Phosphofructo-2-kinase

肝脏双功能酶6-磷酸果糖-2-激酶果糖- 2,6-双磷酸酶已被分离并通过多种生物物理和生化方法进行了表征。PI测定了其蛋白和cDNA序列，并表明该酶由两个独立的结构域组成：nh2末端激酶结构域和cooh末端双磷酸酶结构域。后者在结构和功能上与磷酸甘油酸变化酶/酸性磷酸酶家族同源。最近对2-激酶结构域的结构分析显示其与6-磷酸果糖-1激酶具有结构同源性。计划使用结构信息和生化方法以及位点特异性诱变方法来扩展6PF-2-K催化机制的知识。将研究四个问题：1)基于结构信息，在6-磷酸果糖-2激酶假设的核苷酸结合折叠位点上进行定点诱变，以确认其在ATP结合中的作用。2)确定几个角色；半胱氨酸残基在2-激酶反应中起着重要的作用。3)确定6-PF-2-K反应的特异性。4)基于6PF- 1-K的拓扑结构与预测6PF-2- k改变Fru - 6-P结合残基的拓扑结构的比较。取代的影响将通过测量酶活性、Fru 6-P和ATP亲和力、光谱性质和蛋白质结构来研究。

The liver bifunctional enzyme, 6-phosphofructo-2-kinase fructose- 2,6-bisphosphatase has been isolated and characterized by a number of biophysical and biochemical methods. The PI has determined its protein and cDNA sequence and has shown that the enzyme is composed of two independent domains: a NH2-terminal kinase domain and a COOH-terminal bisphosphatase domain. The latter is structurally and functionally homologous to the phosphoglycerate mutase/acid phosphatase family. Recent structural analysis of the 2-kinase domain has revealed a structural homology with 6-phosphofructo-1-kinases. It is planned to use structural information and biochemical approaches as well as methods of site specific mutagenesis to extend knowledge of the mechanisms involved in 6PF-2-K catalysis. Four questions will be studied: 1) Based on structural information to employ site directed mutagenesis at the site of the hypothesized nucleotide binding fold of 6-phosphofructo-2-kinase in order to confirm its role in ATP binding. 2) To determine the role of several; cysteinyl residues which have been implicated from protein modification experiments as being important in the 2-kinase reaction. 3) To determine the anomeric specificity of the 6-PF-2-K reaction. 4) Based on comparison of the topological structure of 6PF- 1-K and that predicted for 6PF-2-K to alter residues responsible for Fru 6-P binding. The effects of substitutions will be studied by measuring aspects of enzyme activity, Fru 6-P and ATP affinity, spectral properties, and protein structure.