Crystal Studies of Enolase and Transcarboxylase

将结合X射线结晶学、定点突变和动力学研究来研究烯醇化酶的结构和功能关系。每一烯醇化酶亚单位有两个或三个二价金属离子结合位点，它们是按顺序填充的。一些金属离子激活烯醇化酶，另一些金属离子强烈结合但不激活。Lebioda博士将研究烯醇化酶-金属离子-底物-金属离子的三元和四元络合物，以成像酶机制中的连续步骤。将获得突变的烯醇化酶晶体(与布鲁尔和罗布森博士合作)，并确定其结构。对氟离子抑制烯醇化酶的机理进行了研究。单链DNA与烯醇化酶的相互作用将通过研究寡核苷酸与烯醇化酶的复合体来模拟。酵母烯醇化酶晶体分散到1.8A分辨率；这使该系统成为拟议研究的极好对象。除了烯醇化酶的研究，Lebioda博士还建议研究另一种金属酶--转羧酶(TC)的结构。TC与其他生物素酶的一个或多个结构域具有显著的序列一致性，表明TC与其他生物素酶的一个或多个结构域具有同源性。因此，它的结构将是这类蛋白质的代表。

Structure function relationship in enolase will be studied using a combination of x-ray crystallography, site directed mutagenesis, and kinetic studies. There are two, or perhaps three, divalent metal ion binding sites per subunit of enolase which are sequentially filled. Some metal ions activate enolase, others bind strongly but don not activate. Dr. Lebioda will study ternary and quaternary complexes of enolase- metal ion-substrate-metal ion to image the sequential steps in the enzyme mechanism. Crystals of mutant enolase will be obtained (in collaboration with Drs. Brewer and Robson) and their structure determined. The mechanism of enolase inhibition by fluoride ions will be studied. Interaction of single stranded DNA with enolase will be modeled by studying the complexes of oligonucleotides with enolase. The yeast enolase crystals scatter to 1.8A resolution; this makes the system an excellent object of the proposed studies. In addition to enolase research, Dr. Lebioda also proposes to study the structure of another metalloenzyme, transcarboxylase (TC). There is significant sequence identity indicating homology between TC and one or more domains of other biotin enzymes. Thus, its structure will be representative for this class of proteins.