McLaughlin P J, Stuart D I, Klein H W, Oikonomakos N G, Johnson L N
Biochemistry. 1984 Nov 20;23(24):5862-73. doi: 10.1021/bi00319a028.
The structural relationships between substrate and pyridoxal phosphate in glycogen phosphorylase b (EC 2.4.1.1) have been studied by X-ray diffraction experiments at 3-A resolution. Recent work [Klein, H. W., Im, M. J., & Helmreich, E. J. M. (1984) in Chemical and Biological Aspects of Vitamin B6 Catalysis (Evangelopoulos, A. E., Ed.) pp 147-160, Liss, New York] has shown that phosphorylase in the presence of inorganic phosphate catalyzes the conversion of heptenitol to heptulose 2-phosphate. The latter compound is a dead-end product and a most potent inhibitor (Ki = 14 microM). The X-ray diffraction studies show that heptenitol binds at the catalytic site of phosphorylase in a position essentially identical with that observed for the glucopyranose moiety of glucose 1-phosphate. Incubation of a phosphorylase b crystal for 50 h in a solution containing the substrates heptenitol and inorganic phosphate and the activators AMP and maltohetaose resulted in the formation of a phosphorylated product bound at the active site. The structure of this product, as analyzed by a difference Fourier synthesis at 3 A, is consistent with that of heptulose 2-phosphate. Analysis of the surrounding soak solution by thin-layer chromatography showed that heptulose 2-phosphate was produced under these conditions. Heptulose 2-phosphate binds with its glucopyranose moiety in the same position as that for glucose 1-phosphate, but there is a marked difference in phosphate positions. The presence of the methyl group in the beta-configuration in heptulose 2-phosphate forces a change in the torsion angle O5-C1-O1-P from 117 degrees as observe in glucose 1-phosphate to -136 degrees in heptulose 2-phosphate. The "down" position of the phosphate (with respect to the crystallographic z axis) results in a change in the distance between the 5'-phosphorus atom of the pyridoxal phosphate and the phosphorus atom of the substrate from 6.8 (with glucose 1-phosphate) to 4.5 A (with heptulose 2-phosphate). The closest distance between the phosphate oxygen of the cofactor and a phosphate oxygen of heptulose 2-phosphate is 2.7 A, and it is assumed that there must be a hydrogen bond between them. These observations are consistent with the NMR experiments reported in the preceding paper in which sharing of a proton between heptulose 2-phosphate and pyridoxal 5'-phosphate is observed [Klein, H.W., Im, M. J., Palm, D., & Helmreich, E. J. M. (1984) Biochemistry (preceding paper in this issue)].(ABSTRACT TRUNCATED AT 400 WORDS)
已通过3埃分辨率的X射线衍射实验研究了糖原磷酸化酶b（EC 2.4.1.1）中底物与磷酸吡哆醛之间的结构关系。最近的研究工作[Klein, H. W., Im, M. J., & Helmreich, E. J. M.（1984年），载于《维生素B6催化的化学和生物学方面》（Evangelopoulos, A. E.编），第147 - 160页，Liss出版社，纽约]表明，在无机磷酸盐存在下，磷酸化酶催化庚烯醇转化为2 - 磷酸庚酮糖。后一种化合物是一种终产物且是一种极强的抑制剂（Ki = 14微摩尔）。X射线衍射研究表明，庚烯醇在磷酸化酶的催化位点结合，其位置与1 - 磷酸葡萄糖的吡喃葡萄糖部分所观察到的位置基本相同。将磷酸化酶b晶体在含有底物庚烯醇和无机磷酸盐以及激活剂AMP和麦芽庚糖的溶液中孵育50小时，导致在活性位点形成一种结合的磷酸化产物。通过3埃的差值傅里叶合成分析该产物的结构，与2 - 磷酸庚酮糖的结构一致。通过薄层色谱分析周围的浸泡溶液表明，在这些条件下产生了2 - 磷酸庚酮糖。2 - 磷酸庚酮糖的吡喃葡萄糖部分与1 - 磷酸葡萄糖在相同位置结合，但磷酸位置有显著差异。2 - 磷酸庚酮糖中β构型甲基的存在迫使扭转角O5 - C1 - O1 - P从1 - 磷酸葡萄糖中观察到的117度变为2 - 磷酸庚酮糖中的 - 136度。磷酸的“向下”位置（相对于晶体学z轴）导致磷酸吡哆醛的5'-磷原子与底物的磷原子之间的距离从（与1 - 磷酸葡萄糖结合时的）6.8埃变为（与2 - 磷酸庚酮糖结合时的）4.5埃。辅因子的磷酸氧与2 - 磷酸庚酮糖的磷酸氧之间的最短距离为2.7埃，假定它们之间一定存在氢键。这些观察结果与前一篇论文中报道的核磁共振实验一致，在前一篇论文中观察到2 - 磷酸庚酮糖与5'-磷酸吡哆醛之间存在质子共享[Klein, H. W., Im, M. J., Palm, D., & Helmreich, E. J. M.（1984年），《生物化学》（本期前一篇论文）]。（摘要截断于400字）
