Kinetic and mechanistic analysis of Trypanosoma cruzi trans-sialidase reveals a classical ping-pong mechanism with acid/base catalysis
Kinetic and mechanistic analysis of Trypanosoma cruzi trans-sialidase reveals a classical ping-pong mechanism with acid/base catalysis
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DOI：
10.1021/bi7024832
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发表时间：
2008-03-18
期刊：
影响因子：
2.9
通讯作者：
Withers, Stephen G.
中科院分区：
文献类型：
--
作者：
Damager, Iben;Buchini, Sabrina;Withers, Stephen G.
来自克氏锥虫的转唾液酸酶催化唾液酸部分从唾液酸化供体底物转移到乳糖和乳糖苷受体的末端半乳糖部分，以产生α-（2，3）-唾液酸乳糖或其衍生物，其净保留异头构型。通过动力学分析，其中两个不同的供体芳基α-唾液酸苷底物和受体底物乳糖的浓度独立变化，我们已经证明，这种酶遵循乒乓双双动力学机制。这是支持的天然酶和突变体（D59 A），其中假定的酸/碱催化剂已被取代的半反应，其中形成的唾液酸-酶中间体的演示。蛋白质的质谱分析直接证明了共价中间体的形成，而观察到突变体在预稳态爆发中释放完全当量的对硝基苯酚提供了进一步的支持。通过使用D59 A突变体捕获唾液酸酶中间体并对纯化的消化肽进行测序，确认活性位点亲核试剂为Tyr 342。D59作为酸/碱催化剂的作用通过化学拯救研究证实，其中通过叠氮化物恢复D59 A突变体的活性并形成唾液酰叠氮化物产物。
The trans-sialidase from Trypanosoma cruzi catalyzes the transfer of a sialic acid moiety from sialylated donor substrates to the terminal galactose moiety of lactose and lactoside acceptors to yield alpha-(2,3)-sialyllactose or its derivatives with net retention of anomeric configuration. Through kinetic analyses in which the concentrations of two different donor aryl alpha-sialoside substrates and the acceptor substrate lactose were independently varied, we have demonstrated that this enzyme follows a ping-pong bi-bi kinetic mechanism. This is supported for both the native enzyme and a mutant (D59A) in which the putative acid/base catalyst has been replaced by the demonstration of the half-reaction in which a sialyl-enzyme intermediate is formed. Mass spectrometric analysis of the protein directly demonstrates the formation of a covalent intermediate, while the observation of release of a full equivalent of p-nitrophenol by the mutant in a pre-steady state burst provides further support. The active site nucleophile is confirmed to be Tyr342 by trapping of the sialyl-enzyme intermediate using the D59A mutant and sequencing of the purified peptic peptide. The role of D59 as the acid/base catalyst is confirmed by chemical rescue studies in which activity is restored to the D59A mutant by azide and a sialyl azide product is formed.