Wang G P, Cahill S M, Liu X, Girvin M E, Grubmeyer C
Fels Institute for Cancer Research and Molecular Biology, Department of Biochemistry, Temple University School of Medicine, Phildelphia, Pennsylvania 19140, USA.
Biochemistry. 1999 Jan 5;38(1):284-95. doi: 10.1021/bi982057s.
In de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase catalyzes the formation of orotidine 5'-monophosphate (OMP) from orotic acid and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). The known three-dimensional structure of the dimeric enzyme from Salmonella typhimurium is similar to that of other Type I phosphoribosyltransferases (nucleotide synthases) with a solvent-exposed active site atop a Rossman-type nucleotide binding fold. The three-dimensional structure of an enzyme-inhibitor complex [Henriksen et al. (1996) Biochemistry 35, 3803-3809] indicates that one of the two identical solvent-exposed loops can descend to cover the active site of the adjacent subunit of the dimeric enzyme. Catalytically essential residues are known to reside on this loop. In the present work, sensitivity toward limited proteolysis by trypsin confirms that the loop is solvent-exposed. Protection by PRPP and, to a lesser extent, by OMP demonstrates the existence of a second, trypsin-inaccessible, loop position. Two-dimensional 1H-15N NMR relaxation experiments on [alpha-15N]histidine-labeled WT OPRTase yielded backbone 15N T1 and T2 relaxation times and 15N[1H] NOE for His-105 (a loop residue) that are characteristic of small peptides. These results document that the surface loop is highly flexible in the unliganded enzyme. Addition of a hydrolytically stable PRPP analogue to the enzyme resulted in a significant reduction of His-105 peak intensity, indicating a dramatic change in the dynamic properties of the loop backbone in the analogue-ligated enzyme. 1H NMR titrations on histidine C2 protons, coupled with 1H and 31P titrations monitoring the C1H and 5-phosphate PRPP resonances, allowed the quantitation of the rates of loop movement during product release, and relate protein motion to enzymatic catalysis. These results suggest that loop opening and PRPP release is a two-step process, whose overall rate is partially rate-limiting in the reverse pyrophosphorolysis reaction.
在从头嘧啶生物合成过程中，乳清酸磷酸核糖基转移酶催化乳清酸和α-D-5-磷酸核糖-1-焦磷酸（PRPP）生成乳清苷5'-单磷酸（OMP）。鼠伤寒沙门氏菌二聚体酶的已知三维结构与其他I型磷酸核糖基转移酶（核苷酸合酶）相似，其溶剂暴露的活性位点位于罗斯曼型核苷酸结合折叠的顶部。一种酶-抑制剂复合物的三维结构[亨利克森等人（1996年），《生物化学》35卷，3803 - 3809页]表明，两个相同的溶剂暴露环中的一个可以下降以覆盖二聚体酶相邻亚基的活性位点。已知催化必需残基位于该环上。在本研究中，对胰蛋白酶有限蛋白水解的敏感性证实了该环是溶剂暴露的。PRPP以及在较小程度上OMP的保护作用证明存在第二个胰蛋白酶无法接近的环位置。对[α-15N]组氨酸标记的野生型OPRTase进行二维1H - 15N NMR弛豫实验，得到了His - 105（一个环残基）的主链15N T1和T2弛豫时间以及15N[1H] NOE，这些是小肽的特征。这些结果证明表面环在未结合配体的酶中高度灵活。向酶中添加水解稳定的PRPP类似物导致His - 105峰强度显著降低，表明在类似物结合的酶中环主链的动态性质发生了巨大变化。对组氨酸C2质子进行1H NMR滴定，同时结合监测C1H和5 - 磷酸PRPP共振的1H和31P滴定，能够定量产物释放过程中环移动的速率，并将蛋白质运动与酶催化联系起来。这些结果表明环打开和PRPP释放是一个两步过程，其总速率在反向焦磷酸解反应中部分限速。
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