Structure of a Michaelis complex analogue: Propionate binds in the substrate carboxylate site of alanine racemase
Structure of a Michaelis complex analogue: Propionate binds in the substrate carboxylate site of alanine racemase
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DOI：
10.1021/bi9822729
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发表时间：
1999-03-16
期刊：
影响因子：
2.9
通讯作者：
Ringe, D
中科院分区：
文献类型：
--
作者：
Morollo, AA;Petsko, GA;Ringe, D
利用x射线晶体学，以1.9埃的分辨率测定了在活性位点结合丙酸抑制剂的嗜热脂肪芽孢杆菌丙氨酸消旋酶的结构。该酶在溶液中为同二聚体，在不对称单元中与二聚体结晶。两个活性位点都含有一个吡哆醛5′-磷酸（PLP）分子，以醛二胺连接Lys39作为质子化的希夫碱，紫外-可见吸收光谱的ph无关性表明质子化的PLP-Lys39希夫碱是该酶的活性形式。结合在活性位点的丙酸羧酸基团与活性位点残基进行大量相互作用，确定酶的底物结合位点。因此，丙酸结合结构近似于丙氨酸消旋酶与其氨基酸底物之间形成的Michaelis复合体的特征。该结构也提供了在Lys129侧链氨基上形成氨基甲酸酯的证据，通过与丙酸羧酸基Arg136相互作用的残基相互作用来稳定氨基甲酸酯的存在。我们提出这种新的相互作用通过精确定位Arg136和调节其电荷来影响底物结合和催化。
The structure of alanine racemase from Bacillus stearothermophilus with the inhibitor propionate bound in the active site was determined by X-ray crystallography to a resolution of 1.9 Angstrom. The enzyme is a homodimer in solution and crystallizes with a dimer in the asymmetric unit. Both active sites contain a pyridoxal 5'-phosphate (PLP) molecule in aldimine linkage to Lys39 as a protonated Schiff base, and the pH-independence of UV-visible absorption spectra suggests that the protonated PLP-Lys39 Schiff base is the reactive form of the enzyme. The carboxylate group of propionate bound in the active site makes numerous interactions with active-site residues, defining the substrate binding site of the enzyme. The propionate-bound structure therefore approximates features of the Michaelis complex formed between alanine racemase and its amino acid substrate. The structure also provides evidence for the existence of a carbamate formed on the side-chain amino group of Lys129, stabilized by interactions with one of the residues interacting with the carboxylate group of propionate, Arg136. We propose that this novel interaction influences both substrate binding and catalysis by precisely positioning Arg136 and modulating its charge.