Synstad Bjørnar, Gåseidnes Sigrid, Van Aalten Daan M F, Vriend Gert, Nielsen Jens E, Eijsink Vincent G H
Department of Chemistry and Biotechnology, Agricultural University of Norway, As, Norway.
Eur J Biochem. 2004 Jan;271(2):253-62. doi: 10.1046/j.1432-1033.2003.03923.x.
Glycoside hydrolysis by retaining family 18 chitinases involves a catalytic acid (Glu) which is part of a conserved DXDXE sequence motif that spans strand four of a (betaalpha)8 barrel (TIM barrel) structure. These glycoside hydrolases are unusual in that the positive charge emerging on the anomeric carbon after departure of the leaving group is stabilized by the substrate itself (the N-acetyl group of the distorted -1 sugar), rather than by a carboxylate group on the enzyme. We have studied seven conserved residues in the catalytic center of chitinase B from Serratia marcescens. Putative roles for these residues are proposed on the basis of the observed mutational effects, the pH-dependency of these effects, pKa calculations and available structural information. The results indicate that the pKa of the catalytic acid (Glu144) is 'cycled' during catalysis as a consequence of substrate-binding and release and, possibly, by a back and forth movement of Asp142 between Asp140 and Glu144. Rotation of Asp142 towards Glu144 also contributes to an essential distortion of the N-acetyl group of the -1 sugar. Two other conserved residues (Tyr10 and Ser93) are important because they stabilize the charge on Asp140 while Asp142 points towards Glu144. Asp215, lying opposite Glu144 on the other side of the scissile glycosidic bond, contributes to catalysis by promoting distortion of the -1 sugar and by increasing the pKa of the catalytic acid. The hydroxyl group of Tyr214 makes a major contribution to the positioning of the N-acetyl group of the -1 sugar. Taken together, the results show that catalysis in family 18 chitinases depends on a relatively large number of (partly mobile) residues that interact with each other and the substrate.
18家族保留型几丁质酶催化糖苷水解涉及一种催化酸（Glu），它是保守的DXDXE序列基序的一部分，该基序跨越（βα）8桶（TIM桶）结构的第四条链。这些糖苷水解酶的不同寻常之处在于，离去基团离开后在异头碳上出现的正电荷由底物本身（扭曲的-1糖的N-乙酰基）稳定，而不是由酶上的羧酸盐基团稳定。我们研究了粘质沙雷氏菌几丁质酶B催化中心的七个保守残基。根据观察到的突变效应、这些效应的pH依赖性、pKa计算和可用的结构信息，对这些残基的假定作用进行了推测。结果表明，催化酸（Glu144）的pKa在催化过程中由于底物结合和释放而“循环”，并且可能是由于Asp142在Asp140和Glu144之间来回移动。Asp142向Glu144的旋转也有助于-1糖的N-乙酰基发生必要的扭曲。另外两个保守残基（Tyr10和Ser93）很重要，因为当Asp142指向Glu144时，它们能稳定Asp140上的电荷。位于可裂解糖苷键另一侧与Glu144相对的Asp215，通过促进-1糖的扭曲和提高催化酸的pKa来促进催化作用。Tyr214的羟基对-1糖的N-乙酰基的定位起主要作用。综上所述，结果表明18家族几丁质酶的催化作用依赖于相对大量的（部分可移动的）相互作用以及与底物相互作用的残基。
