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- 已分离出2，6-二磷酸酶，并通过 一些生物物理和生物化学方法。 主要研究者有 确定了它的蛋白质和cDNA序列，并表明， 酶由两个独立的结构域组成： 激酶结构域和COOH-末端双磷酸酶结构域。 的 后者在结构和功能上与 磷酸甘油酸酯/酸性磷酸酶家族。 最近 2-激酶结构域的结构分析揭示了 与6-磷酸果糖-1-激酶结构同源。 是 计划使用结构信息和生物化学方法 以及位点特异性诱变的方法， 了解6PF-2-K催化的机制。 四 将研究的问题： 1)根据结构信息，采用现场指导 在假设的核苷酸结合位点的诱变 6-磷酸果糖-2-激酶的折叠，以确认其在 ATP结合。 2)确定几个半胱氨酸残基的作用 蛋白质修饰实验表明 在2-激酶反应中很重要。 3)确定6-PF-2-K的异头特异性 反应 4)在比较了6PF- 1-K和预测的6PF-2-K改变残基负责 用于Fru 6-P结合。 替代的影响将通过测量来研究。 酶活性、Fru 6-P和ATP亲和力、光谱 性质和蛋白质结构。

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.