Pummill Philip E, DeAngelis Paul L
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
J Biol Chem. 2002 Jun 14;277(24):21610-6. doi: 10.1074/jbc.M202456200. Epub 2002 Apr 9.
The hyaluronan (HA) synthases catalyze the addition of two different monosaccharides from UDP-sugar substrates to the linear heteropolysaccharide chain. To accomplish this task, the HA synthases must be able to bind and to transfer from both UDP-sugar substrates. Until now, it has been impossible to distinguish between these two abilities. We have created a mutant of xlHAS1, a HA synthase from Xenopus laevis, that allows for the examination of the enzyme's ability to bind substrate only. The ability of different compounds to protect the xlHAS1(C337S) mutant enzyme from loss of activity due to treatment with N-ethylmaleimide, a cysteine-modifying reagent, yields information on the relative affinity of a variety of nucleotides and nucleotide-sugars. We have observed that the substrate binding selectivity is more relaxed than the specificity of catalytic transfer. The only attribute that appears to be absolutely required for binding is a nucleotide containing two phosphates complexed with magnesium ion. The role of certain cysteine residues in catalysis was also evaluated. Cys307 of xlHAS1 may play a role in catalysis or in maintaining structure. Mutation of Cys337 raises the UDP-GlcUA Michaelis constant (K(m)), suggesting that this residue participates in UDP-GlcUA substrate binding or in catalytic complex formation.
透明质酸（HA）合酶催化将来自UDP-糖底物的两种不同单糖添加到线性杂多糖链上。为完成此任务，HA合酶必须能够结合并从两种UDP-糖底物进行转移。到目前为止，还无法区分这两种能力。我们构建了非洲爪蟾的HA合酶xlHAS1的一个突变体，该突变体仅允许检测该酶结合底物的能力。不同化合物保护xlHAS1（C337S）突变体酶免受半胱氨酸修饰试剂N-乙基马来酰亚胺处理导致的活性丧失的能力，产生了关于多种核苷酸和核苷酸糖相对亲和力的信息。我们观察到底物结合选择性比催化转移的特异性更宽松。结合似乎绝对必需的唯一属性是含有与镁离子络合的两个磷酸基团的核苷酸。还评估了某些半胱氨酸残基在催化中的作用。xlHAS1的Cys30７可能在催化或维持结构中起作用。Cys337的突变提高了UDP-GlcUA米氏常数（Kｍ），表明该残基参与UDP-GlcUA底物结合或催化复合物形成。
