Beattie B K, Prentice G A, Merrill A R
Guelph-Waterloo Centre for Graduate Work in Chemistry, Ontario Department of Chemistry and Biochemistry, University of Guelph, Canada.
Biochemistry. 1996 Dec 3;35(48):15134-42. doi: 10.1021/bi961985t.
The role of the tryptophan residues in the substrate-binding and catalytic mechanism of an enzymatically active C-terminal fragment of Pseudomonas aeruginosa exotoxin A was studied by individually or jointly replacing these residues with phenylalanine. Substitution of W-466 decreased the ADP-ribosyltransferase and NAD(+)-glycohydrolase activities by 20- and 3-fold, respectively. In contrast, substitution of W-417 or W-558 with phenylalanine both resulted in a 3-fold decrease in ADP-ribosyltransferase activity with, however, only a decrease by 40% and 70% in NAD(+)-glycohydrolase activity, respectively. Simultaneous replacement of W-466 and W-558 resulted in a 200-fold decrease in ADP-ribosyltransferase and an 6-fold decrease in NAD(+)-glycohydrolase activities, suggesting that W-466 may play a minor role in the transfer of ADP-ribose to the eEF-2 protein. Chemical modification of the tryptophan residues in the wild-type toxin fragment by N-bromosuccinimide revealed the presence of a single residue important for enzymatic activity, W-466, with a minor contribution from W-558. Additionally, tryptophan residues, W-305 and W-417, were refractory to oxidation by N-bromosuccinimide, which likely indicated the buried nature of these residues within the protein structure. Titration of the wild-type toxin fragment with NAD+ resulted in the quenching of the intrinsic tryptophan fluorescence to 58% of the initial value. Titration of the various single and a double tryptophan replacement mutant protein(s) indicated that W-558 and W-466 are responsible for the substrate-induced fluorescence quenching, with the former being responsible for the largest fraction of the observed quenching in the wild-type toxin. Consequently, a molecular mechanism is proposed for the substrate-induced fluorescence quenching of both W-466 and W-558. Furthermore, molecular modeling of the recent crystal structures for both exotoxin A (domain III fragment) and diphtheria toxin, combined with a variety of previous results, has led to the proposal for a catalytic mechanism for the ADP-ribosyltransferase reaction. This mechanism features a SN1 attack (instead of the previously purported SN2 mechanism) by the diphthamide residue (nucleophile) of eukaryotic elongation factor 2 on the C-1 of the nicotinamide ribose of NAD+, which results in an inversion of configuration likely due to steric constraints within the NAD(+)-toxin-elongation factor 2 complex.
通过将色氨酸残基单独或联合替换为苯丙氨酸，研究了色氨酸残基在铜绿假单胞菌外毒素A酶活性C末端片段的底物结合和催化机制中的作用。W-466被替换后，ADP-核糖基转移酶和NAD(+) - 糖水解酶活性分别降低了20倍和3倍。相比之下，用苯丙氨酸替换W-417或W-558，ADP-核糖基转移酶活性均降低了3倍，然而，NAD(+) - 糖水解酶活性仅分别降低了40%和70%。同时替换W-466和W-558导致ADP-核糖基转移酶活性降低200倍，NAD(+) - 糖水解酶活性降低6倍，这表明W-466在将ADP-核糖转移到eEF-2蛋白的过程中可能起次要作用。用N-溴代琥珀酰亚胺对野生型毒素片段中的色氨酸残基进行化学修饰，发现存在一个对酶活性重要的单一残基W-466，W-558的贡献较小。此外，色氨酸残基W-305和W-417对N-溴代琥珀酰亚胺的氧化具有抗性，这可能表明这些残基在蛋白质结构中是埋藏的。用NAD+滴定野生型毒素片段导致内在色氨酸荧光猝灭至初始值的58%。对各种单和双色氨酸替换突变蛋白的滴定表明，W-558和W-466负责底物诱导的荧光猝灭，前者在野生型毒素中观察到的猝灭中占最大比例。因此，提出了W-466和W-558底物诱导荧光猝灭的分子机制。此外，外毒素A（结构域III片段）和白喉毒素最近晶体结构的分子建模，结合各种先前的结果，导致了ADP-核糖基转移酶反应催化机制的提出。该机制的特点是真核延伸因子2的二肽酰胺残基（亲核试剂）对NAD+烟酰胺核糖C-1进行SN1攻击（而不是先前推测的SN2机制），这可能由于NAD(+) - 毒素 - 延伸因子2复合物内的空间限制导致构型反转。
