Morollo A A, Petsko G A, Ringe D
Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254, USA.
Biochemistry. 1999 Mar 16;38(11):3293-301. doi: 10.1021/bi9822729.
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 A. 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.
通过X射线晶体学确定了嗜热脂肪芽孢杆菌丙氨酸消旋酶的结构，其活性位点结合有抑制剂丙酸，分辨率为1.9埃。该酶在溶液中是同型二聚体，在不对称单元中以二聚体形式结晶。两个活性位点都含有一个与Lys39以质子化席夫碱形式形成醛亚胺键的磷酸吡哆醛（PLP）分子，紫外可见吸收光谱的pH独立性表明质子化的PLP-Lys39席夫碱是酶的活性形式。结合在活性位点的丙酸的羧基与活性位点残基发生大量相互作用，确定了酶的底物结合位点。因此，结合丙酸的结构近似于丙氨酸消旋酶与其氨基酸底物形成的米氏复合物的特征。该结构还为在Lys129侧链氨基上形成的氨基甲酸酯的存在提供了证据，该氨基甲酸酯通过与与丙酸羧基相互作用的残基之一Arg136的相互作用而稳定。我们提出，这种新的相互作用通过精确地定位Arg136并调节其电荷来影响底物结合和催化作用。
