MECHANISMS OF NUCLEOTIDE SUGAR INTERCONVERSIONS

大肠杆菌半乳糖-1-P尿苷酰转移酶和 UDP-半乳糖4-差向异构酶的特征在于， 鉴定参与催化或结合的氨基酸官能团 的substrates。 开发新技术将被优先考虑 用于标记和鉴定核苷酸结合位点的酶基团。 结合在活性位点的核苷酸的硫类似物将被激活， 与亲电试剂反应以将硫核苷酸转化为 用于磷酸化活性位点残基的活性磷酸化剂。 32 P标记的蛋白质将被降解为磷酸化氨基酸以鉴定 磷酸化位点。 这种方法将得到补充， 基团选择性试剂和活性位点导向的烷基化试剂， 修饰其他官能团。 任何活性物质的催化功能 将在研究中进一步研究确定的位点残基， 反应动力学 V、Km和V/Km的pH依赖性，使用两种正常 并且缓慢反应的底物将被确定。 预计在 对于缓慢反应的底物，产物解离步骤将不受限制 速率，并且在这些条件下，pH速率数据可以给出有价值的 活性位点中催化基团的pKa值信息 这些酶。 将测量同位素效应和交换率， 确定产品解离步骤是否部分速率 限制慢衬底。

The active sites of Escherichia coli galactose-1-P uridylyltransferase and UDP-galactose 4-epimerase will be characterized with the objective of identifying amino acid functional groups involved in catalysis or binding of substrates. First priority will be given to developing a new technique for labeling and identifying enzymic groups at nucleotide binding sites. Sulfur analogs of nucleotides bound at active sites will be activated by reaction with electrophilic reagents to convert the sulfur nucleotides into active phosphorylating agents for phosphorylating active site residues. The 32P-labeled protein will be degraded to phospho amino acids to identify the phosphorylation sites. This approach will be complemented by the use of group-selective reagents and active site-directed alkylating agents to modify other functional groups. The catalytic functions of any active site-residues identified will be further investigated in studies of the reaction kinetics. The pH-dependence of V, Km and V/Km using both normal and slowly reacting substrates will be determined. It is expected that with slowly reacting substrates product dissociation step will not limit rates, and under these conditions the pH-rate data may give valuable information about the pKa values for catalytic groups in the active sites of these enzymes. Isotope effects and exchange rates will be measured to determine whether product dissociation steps are or are not partially rate limiting for slow substrates.