Lukac M, Collier R J
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts.
Biochemistry. 1988 Oct 4;27(20):7629-32. doi: 10.1021/bi00420a009.
Directed mutagenesis was used to probe the functions of Tyr-470 and Tyr-481 of Pseudomonas aeruginosa exotoxin A (ETA) with respect to cytotoxicity, ADP-ribosylation of elongation factor 2 (EF-2), and NAD-glycohydrolase activity. Both of these residues lie in the active site cleft, close to Glu-553, a residue believed to play a direct role in catalysis of ADP-ribosylation of EF-2. Substitution of Tyr-470 with Phe caused no change in any of these activities, thus eliminating the possibility that the phenolic hydroxyl group of Tyr-470 might be directly involved in catalysis. Mutation of Tyr-481 to Phe caused an approximately 10-fold reduction in NAD:EF-2 ADP-ribosyltransferase activity and cytotoxicity but no change in NAD-glycohydrolase activity. The latter mutation did not alter the KM of NAD in the NAD-glycohydrolase reaction, which suggests that the phenolic hydroxyl of Tyr-481 does not participate in NAD binding. We hypothesize that the phenolic hydroxyl of Tyr-481 may be involved in the interaction of the toxin with substrate EF-2.
利用定向诱变技术，探究铜绿假单胞菌外毒素A（ETA）的酪氨酸-470（Tyr-470）和酪氨酸-481（Tyr-481）在细胞毒性、延伸因子2（EF-2）的ADP-核糖基化以及NAD-糖水解酶活性方面的功能。这两个残基均位于活性位点裂隙中，靠近谷氨酸-553（Glu-553），据信该残基在EF-2的ADP-核糖基化催化中起直接作用。用苯丙氨酸（Phe）取代Tyr-470后，这些活性均未发生变化，因此排除了Tyr-470的酚羟基可能直接参与催化的可能性。将Tyr-481突变为Phe导致NAD：EF-2 ADP-核糖基转移酶活性和细胞毒性降低约10倍，但NAD-糖水解酶活性未发生变化。后一种突变并未改变NAD-糖水解酶反应中NAD的米氏常数（KM），这表明Tyr-481的酚羟基不参与NAD结合。我们推测，Tyr-481的酚羟基可能参与毒素与底物EF-2的相互作用。
