Evaluation of critical structural elements of UDP-sugar substrates and certain cysteine residues of a vertebrate hyaluronan synthase.
Evaluation of critical structural elements of UDP-sugar substrates and certain cysteine residues of a vertebrate hyaluronan synthase.
复制标题
脊椎动物乙酰透明质酸合酶的 UDP 糖底物和某些半胱氨酸残基的关键结构元件的评估。
DOI：
10.1074/jbc.m202456200
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
DeAngelis,PaulL
中科院分区：
文献类型：
--
作者：
Pummill,PhilipE;DeAngelis,PaulL
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 fromXenopus 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. Cys307of xlHAS1 may play a role in catalysis or in maintaining structure. Mutation of Cys337raises the UDP-GlcUA Michaelis constant (Km), suggesting that this residue participates in UDP-GlcUA substrate binding or in catalytic complex formation.
