Datta A K, Sinha A, Paulson J C
Cytel Corporation and the Department of Chemistry and Molecular Biology, Scripps Research Institute, San Diego, California 92121, USA.
J Biol Chem. 1998 Apr 17;273(16):9608-14. doi: 10.1074/jbc.273.16.9608.
Protein sequence analysis of the cloned sialyltransferase gene family has revealed the presence of two conserved protein motifs in the middle of the lumenal catalytic domain, termed L-sialylmotif and S-sialylmotif. In our previous study (Datta, A. K., and Paulson, J. C. (1995) J. Biol. Chem. 270, 1497-1500) the larger L-sialylmotif of ST6Gal I was analyzed by site-directed mutagenesis, which provided evidence that it participates in the binding of the CMP-NeuAc, a common donor substrate for all the sialyltransferases. However, none of the mutants tested in this motif had any significant effect on their binding affinities toward the acceptor substrate asialo alpha1-acid glycoprotein. In this study, we have investigated the role of the S-sialylmotif of the same enzyme ST6Gal I. In total, nine mutants have been constructed by changing the conserved amino acids of this motif to mostly alanine by site-directed mutagenesis. Kinetic analysis for the mutants which retained sialyltransferase activity showed that the mutations in the S-sialylmotif caused a change of Km values for both the donor and the acceptor substrates. Our results indicated that this motif participates in the binding of both the substrates. A sequence homology search also supported this finding, which showed that the downstream amino acid sequence of the S-sialylmotif is conserved for each subgroup of this enzyme family, indicating its association with the acceptor substrate.
对克隆的唾液酸转移酶基因家族进行的蛋白质序列分析表明，在腔催化结构域中部存在两个保守的蛋白质基序，分别称为L-唾液酸基序和S-唾液酸基序。在我们之前的研究中（达塔，A.K.，和保尔森，J.C.（1995年）《生物化学杂志》270卷，1497 - 1500页），通过定点诱变分析了ST6Gal I较大的L-唾液酸基序，结果表明它参与了CMP-唾液酸（所有唾液酸转移酶的常见供体底物）的结合。然而，在该基序中测试的任何突变体对其与受体底物去唾液酸α1-酸性糖蛋白的结合亲和力都没有显著影响。在本研究中，我们研究了同一种酶ST6Gal I的S-唾液酸基序的作用。通过定点诱变，总共构建了九个突变体，将该基序中的保守氨基酸大多替换为丙氨酸。对保留唾液酸转移酶活性的突变体进行动力学分析表明，S-唾液酸基序中的突变导致供体和受体底物的Km值都发生了变化。我们的结果表明，该基序参与了两种底物的结合。序列同源性搜索也支持了这一发现，结果表明该酶家族每个亚组的S-唾液酸基序下游氨基酸序列是保守的，表明它与受体底物相关。
