Lavie A, Allen K N, Petsko G A, Ringe D
Department of Biochemistry, Brandeis University, Massachusetts 02254-9110.
Biochemistry. 1994 May 10;33(18):5469-80. doi: 10.1021/bi00184a016.
The X-ray crystallographic structures of the metal-activated enzyme xylose isomerase from Streptomyces olivochromogenes with the substrates D-glucose, 3-O-methyl-D-glucose and in the absence of substrate were determined to 1.96-, 2.19-, and 1.81-A resolution and refined to R-factors of 16.6%, 15.9%, and 16.1%, respectively. Xylose isomerase catalyzes the interconversion between glucose and fructose (xylose and xylulose under physiological conditions) by utilizing two metal cofactors to promote a hydride shift; the metals are bridged by a glutamate residue. This puts xylose isomerase in the small but rapidly growing family of enzymes with a bridged bimetallic active site, in which both metals are involved in the chemical transformation. The substrate 3-O-methylglucose was chosen in order to position the glucose molecule in the observed electron density unambiguously. Of the two essential magnesium ions per active site, Mg-2 was observed to occupy two alternate positions, separated by 1.8 A, in the substrate-soaked structures. The deduced movement was not observed in the structure without substrate present and is attributed to a step following substrate binding but prior to isomerization. The substrates glucose and 3-O-methylglucose are observed in their linear extended forms and make identical interactions with the enzyme by forming ligands to Mg-1 through O2 and O4 and by forming hydrogen bonds with His53 through O5 and Lys182 through O1. Mg-2 has a water ligand that is interpreted in the crystal structure in the absence of substrate as a hydroxide ion and in the presence of substrate as a water molecule. This hydroxide ion may act as a base to deprotonate the glucose O2 and subsequently protonate the product fructose O1 concomitant with hydride transfer. Calculations of the solvent-accessible surface of possible dimers, with and without the alpha-helical C-terminal domain, suggest that the tetramer is the active form of this xylose isomerase.
测定了来自橄榄色链霉菌的金属激活酶木糖异构酶在存在底物D-葡萄糖、3-O-甲基-D-葡萄糖以及不存在底物情况下的X射线晶体结构，分辨率分别为1.96 Å、2.19 Å和1.81 Å，并分别精修至R因子为16.6%、15.9%和16.1%。木糖异构酶通过利用两个金属辅因子促进氢化物转移来催化葡萄糖和果糖之间的相互转化（在生理条件下为木糖和木酮糖）；金属由一个谷氨酸残基桥连。这使木糖异构酶属于具有桥连双金属活性位点的酶的小而快速增长的家族，其中两种金属都参与化学转化。选择底物3-O-甲基葡萄糖是为了明确地将葡萄糖分子定位在观察到的电子密度中。在每个活性位点的两个必需镁离子中，观察到Mg-2在底物浸泡的结构中占据两个交替位置，相隔1.8 Å。在不存在底物的结构中未观察到推断的移动，这归因于底物结合后但异构化之前的一个步骤。观察到底物葡萄糖和3-O-甲基葡萄糖呈线性伸展形式，并通过O2和O4与Mg-1形成配体以及通过O5与His53和通过O1与Lys182形成氢键与酶进行相同的相互作用。Mg-2有一个水配体，在不存在底物的晶体结构中被解释为氢氧根离子，在存在底物时被解释为水分子。这个氢氧根离子可能作为碱使葡萄糖O2去质子化，随后使产物果糖O1质子化并伴随氢化物转移。对有和没有α-螺旋C末端结构域的可能二聚体的溶剂可及表面的计算表明四聚体是这种木糖异构酶的活性形式。
