SITE DIRECTED SPIN LABELING STUDY OF ACTIVE SITE OF PHOSPHORIBULOKINASE

我们已经使用了定点定向自旋标记来证明活性位点 是卡尔文循环中的一种酶，催化 磷酰基从三磷酸腺苷到5-磷酸核酮糖的转移。 为此，我们产生了两个单半胱氨酸突变体A16C和 K53C通过定点突变，在PrK的活性部位。两个人 对突变体的活性进行了表征，并检查了 使用过滤、CD和滴定的结构微扰 带有自旋标记的ATP探针的活性部位，ATPSAP。变种人展示了 与Wild Type没有明显的偏差。然后给它们贴上自旋标签 在存在和不存在的情况下记录了MTSSL及其光谱 ATP和变构效应NADH。SL-K53C光谱没有 A16C谱对ATP或NADH敏感。 后者是预期的，因为A16是 已知的核苷酸结合部位的P-环。实验 正在对双突变A16C/K53C进行研究，以确定距离 在活动站点中。

We have used site-directed spin labeling to prove the active site of phosphoribulokinase, an enzyme in the Calvin cycle that catalyzes the transfer of a phosphoryl group from ATP to ribulose 5-phosohate. To this end, we have generated two single cysteine mutants A16C and k53C by site directed mutagenesis, in the active site of PRK. The two mutants were characterized for activity and also checked for structural perturbation using filtration, CD and titration of the active site with the spin labeled ATP probe, ATPSAP. The mutants show no noticeable deviation from Wild Type. They were then spin labeled with MTSSL and their spectra recorded in the presence and absence of ATP and the allosteric effector NADH. SL-K53C spectrum was not sensitive to ATP or NADH while the A16C spectrum was sensitive to ATP. The latter is expected due to the fact that A16 is a residue in the P-loop which is known to be the nucleotide binding site. Experiments are in progress on the double mutant A16C/K53C to determine distances in the active site.