SITE DIRECTED SPIN LABELING STUDY OF ACTIVE SITE OF PHOSPHORIBULOKINASE

磷酸核糖激酶活性位点的定点自旋标记研究

• 批准号: 6307890
• 负责人: HANANE KOTEICHE
• 金额: $ 1.13万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6307890
• 关键词: bioengineering /biomedical engineering; biological products; biomedical resource; enzymes

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.

我们已经用定点自旋标记来证明活性位点 磷酸核酮糖激酶，一种卡尔文循环中的酶， 将磷酰基从ATP转移到核酮糖5-磷酸。 为此，我们已经产生了两个单半胱氨酸突变体A16 C和 k53 C通过定点突变，在活性位点的p53。 两 对突变体进行了活性表征，并检查了 使用过滤，CD和滴定的结构扰动 活性位点与自旋标记的ATP探针，ATPSAP。 变种人表现出 与野生型无明显差异。 然后他们被自旋标记 与MTSSL和他们的光谱记录在存在和不存在的 ATP和变构效应物NADH。 SL-K53 C光谱未显示 A16 C谱对ATP敏感。 后者是预期的，这是由于A16是化合物中的残基的事实。 P-环，已知是核苷酸结合位点。 实验 正在对双突变体A16 C/K53 C进行研究，以确定 在活动现场。