Strynadka N C, James M N
Department of Biochemistry, University of Alberta, Edmonton, Canada.
J Mol Biol. 1991 Jul 20;220(2):401-24. doi: 10.1016/0022-2836(91)90021-w.
A structure of the trisaccharide 2-acetamido-2-deoxy-D-muramic acid-beta (1----4)-2-acetamido-2-deoxy-D-glucose-beta (1----4)-2-acetamido-2-deoxy-D-muramic acid (NAM-NAG-NAM), bound to subsites B, C and D in the active-site cleft of hen egg-white lysozyme has been determined and refined at 1.5 A resolution. The resulting atomic co-ordinates indicate that the NAM residue in site D is distorted from the full 4C1 chair conformation to one in which the ring atoms C-1, C-2, O-5 and C-5 are approximately coplanar, and the hydroxymethyl group is positioned axially (a conformation best described as a sofa). This finding supports the original proposals that suggested the ground-state conformation of the sugar bound in site D is strained to one that more closely resembles the geometry required for the oxocarbonium-ion transition state, the next step along the reaction pathway. Additionally, detailed analysis at 1.5 A resolution of the environments of the catalytic residues Glu35 and Asp52 provides new information on the properties that may allow lysozyme to promote the stabilization of an unusually long-lived oxocarbonium-ion transition state. Intermolecular interactions between the N-acetylmuramic acid residue in site D and the lysozyme molecule that contribute to the saccharide ring distortion include: close packing of the O-3' lactyl group with a hydrogen-bonded "platform" of enzyme residues (Asp52, Asn46, Asn59, Ser50 and Asp48), a close contact between the hydroxymethyl group of ring D and the 2'-acetamido group of ring C and a strong hydrogen-bonded interaction between the NH group of Val109 and O-6 of ring D that stabilizes the observed quasi-axial orientation of the -CH2OH group. Additionally, the structure of this complex shows a strong hydrogen bond between the carboxyl group of Glu35 and the beta-anomeric hydroxyl group of the NAM residue in site D. The hydrogen-bonded environment of Asp52 in the native enzyme and in the complex coupled with the very unfavorable direction of approach of the potential carboxylate nucleophile makes it most unlikely that there is a covalent glycosylenzyme intermediate on the hydrolysis pathway of hen egg-white lysozyme.
已确定并以1.5埃分辨率精修了三糖2-乙酰氨基-2-脱氧-D-胞壁酸-β(1→4)-2-乙酰氨基-2-脱氧-D-葡萄糖-β(1→4)-2-乙酰氨基-2-脱氧-D-胞壁酸（NAM-NAG-NAM）与鸡蛋清溶菌酶活性位点裂隙中的B、C和D亚位点结合的结构。所得原子坐标表明，位点D中的NAM残基从完整的4C1椅式构象扭曲为环原子C-1、C-2、O-5和C-5大致共面且羟甲基轴向定位的构象（一种最好描述为沙发的构象）。这一发现支持了最初的提议，即位点D中结合的糖的基态构象被拉伸为更类似于氧鎓离子过渡态所需几何结构的构象，这是反应途径中的下一步。此外，以1.5埃分辨率对催化残基Glu35和Asp52的环境进行的详细分析提供了关于可能使溶菌酶促进异常长寿命氧鎓离子过渡态稳定的性质的新信息。位点D中的N-乙酰胞壁酸残基与溶菌酶分子之间有助于糖环扭曲的分子间相互作用包括：O-3'乳酰基与酶残基（Asp52、Asn46、Asn59、Ser50和Asp48）的氢键“平台”紧密堆积，环D的羟甲基与环C的2'-乙酰氨基之间的紧密接触，以及Val109的NH基团与环D的O-6之间稳定观察到的-CH2OH基团准轴向取向的强氢键相互作用。此外，该复合物的结构显示Glu35的羧基与位点D中NAM残基的β-异头羟基之间有强氢键。天然酶和复合物中Asp52的氢键环境，加上潜在羧酸盐亲核试剂非常不利的接近方向，使得鸡蛋清溶菌酶水解途径上极不可能存在共价糖基化酶中间体。
