Blanke S R, Huang K, Collier R J
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts.
Biochemistry. 1994 Dec 27;33(51):15494-500. doi: 10.1021/bi00255a031.
Previous studies have suggested that tyrosine-65 (Tyr-65) of diphtheria toxin (DT) is located at the active site. To investigate the role of Tyr-65 in NAD binding and the ADP-ribosylation of elongation factor-2 (EF-2), we changed this residue to alanine and phenylalanine by site-directed mutagenesis of a synthetic gene encoding the catalytic fragment of DT (DTA). The alanine mutant was greatly diminished in ADP-ribosylation activity (350-fold) and NAD-glycohydrolase activity (88-fold), whereas the phenylalanine mutant was reduced in these activities only slightly. Dissociation constants (Kd) for NAD binding were 15 microM for wild-type DTA, 26 microM for the phenylalanine mutant, and greater than 800 microM NAD for the alanine mutant. However, both mutant enzymes were found to bind adenosine with nearly equal affinity as wild-type DTA. These results support a model of ADP-ribosylation in which the phenolic ring of Tyr-65 interacts with the nicotinamide ring of NAD, orienting the N-glycosidic bond of NAD for attack by the incoming nucleophile in a direct displacement mechanism.
先前的研究表明，白喉毒素（DT）的酪氨酸65（Tyr-65）位于活性位点。为了研究Tyr-65在NAD结合及延伸因子2（EF-2）的ADP核糖基化中的作用，我们通过对编码DT催化片段（DTA）的合成基因进行定点诱变，将该残基分别替换为丙氨酸和苯丙氨酸。丙氨酸突变体的ADP核糖基化活性（降低350倍）和NAD糖水解酶活性（降低88倍）大幅下降，而苯丙氨酸突变体的这些活性仅略有降低。野生型DTA与NAD结合的解离常数（Kd）为15微摩尔，苯丙氨酸突变体为26微摩尔，丙氨酸突变体大于800微摩尔NAD。然而，发现两种突变酶与腺苷的结合亲和力与野生型DTA几乎相等。这些结果支持了一种ADP核糖基化模型，即Tyr-65的酚环与NAD的烟酰胺环相互作用，使NAD的N-糖苷键定向，以便在直接取代机制中被进入的亲核试剂攻击。
