Watts Andrew G, Damager Iben, Amaya Maria L, Buschiazzo Alejandro, Alzari Pedro, Frasch Alberto C, Withers Stephen G
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, B.C., Canada V6T 1Z1.
J Am Chem Soc. 2003 Jun 25;125(25):7532-3. doi: 10.1021/ja0344967.
Modified sialic acid substrates have been used to label Trypanosoma cruzi trans-sialidase, demonstrating that the enzyme catalyses the transfer of sialic acid through a covalent glycosyl-enzyme intermediate, a mechanism common to most retaining glycosidases. Peptic digestion of labeled protein, followed by LC-MS/MS analysis of the digest, identified Tyr342 as the catalytic nucleophile. This is the first such example of a retaining glycosidase utilizing an aryl glycoside intermediate. It is suggested that this alternative choice of nucleophile is a consequence of the chemical nature of sialic acid. A Tyr/Glu couple is invoked to relay charge from a remote glutamic acid, thereby avoiding electrostatic repulsion with the sialic acid carboxylate group.
修饰的唾液酸底物已被用于标记克氏锥虫转唾液酸酶，证明该酶通过共价糖基 - 酶中间体催化唾液酸的转移，这是大多数保留型糖苷酶共有的机制。对标记蛋白进行胃蛋白酶消化，然后对消化产物进行液相色谱 - 串联质谱分析，确定Tyr342为催化亲核试剂。这是利用芳基糖苷中间体的保留型糖苷酶的首个此类例子。有人认为，这种亲核试剂的替代选择是唾液酸化学性质的结果。引入了一个Tyr / Glu对来传递来自远处谷氨酸的电荷，从而避免与唾液酸羧酸盐基团产生静电排斥。
