White Ellen M, Holland Amanda R, MacDonald Gina
Department of Chemistry, James Madison University, Harrisonburg, Virginia 22807, USA.
Biochemistry. 2008 Jan 8;47(1):84-91. doi: 10.1021/bi701723c. Epub 2007 Dec 14.
Phosphoglycerate kinase (PGK) catalyzes a reversible phospho-transfer reaction between ATP and 3-phosphoglycerate (3-PG) that is thought to require a hinge-bending motion in the protein that brings two separate substrate-binding domains together. We have used difference infrared spectroscopy to better understand the conformational changes that are unique to the PGK-ATP-3-PG complex. Caged nucleotides (caged-ADP and caged-ATP) were used to initiate nucleotide binding to PGK or PGK-3-PG complexes. The difference spectra include those of PGK-ATP minus PGK, PGK-3-PG-ATP minus PGK-3-PG, PGK-3-PG-ADP minus PGK-3-PG, and PGK-ADP minus PGK. The resulting spectra were compared in attempts to identify bands associated with each PGK complex. In addition, complementary activity assays were performed in the presence of caged-nucleotides. While PGK activity decreased in the presence of caged-ADP, the activity was not influenced by the addition of caged-ATP. The activity assay results suggest that the caged-ADP may interact with PGK substrate binding site(s) and inhibit phospho-transfer. Therefore, additional difference infrared nucleotide exchange experiments were used to isolate the differences between ADP and ATP binding to PGK. Difference FTIR spectra obtained on PGK-nucleotide-3-PG complexes show distinct bands that may result from amino acid side chains as well as structural changes in the hinge region and/or increased interactions such as salt bridges forming between the two domains. The infrared data obtained on the active ternary complexes show evidence of changes in alpha-helix and beta-structures as well as signals consistent with Arg, Asn, His, Lys, Asp, Glu, and additional side chains that are uniquely perturbed in the active ternary complex as compared to other PGK complexes.
磷酸甘油酸激酶（PGK）催化ATP与3-磷酸甘油酸（3-PG）之间的可逆磷酸转移反应，据认为该反应需要蛋白质中的铰链弯曲运动，使两个独立的底物结合结构域聚集在一起。我们使用差示红外光谱法来更好地理解PGK-ATP-3-PG复合物特有的构象变化。笼形核苷酸（笼形ADP和笼形ATP）用于启动核苷酸与PGK或PGK-3-PG复合物的结合。差示光谱包括PGK-ATP减去PGK、PGK-3-PG-ATP减去PGK-3-PG、PGK-3-PG-ADP减去PGK-3-PG以及PGK-ADP减去PGK的光谱。对所得光谱进行比较，试图识别与每种PGK复合物相关的谱带。此外，在笼形核苷酸存在的情况下进行了互补活性测定。虽然在笼形ADP存在下PGK活性降低，但添加笼形ATP对活性没有影响。活性测定结果表明，笼形ADP可能与PGK底物结合位点相互作用并抑制磷酸转移。因此，使用额外的差示红外核苷酸交换实验来分离ADP和ATP与PGK结合之间的差异。在PGK-核苷酸-3-PG复合物上获得的差示傅里叶变换红外光谱显示出不同的谱带，这些谱带可能源于氨基酸侧链以及铰链区域的结构变化和/或增加的相互作用，如两个结构域之间形成的盐桥。在活性三元复合物上获得的红外数据显示了α螺旋和β结构的变化证据，以及与精氨酸、天冬酰胺、组氨酸、赖氨酸、天冬氨酸、谷氨酸和其他侧链一致的信号，与其他PGK复合物相比，这些侧链在活性三元复合物中受到独特的扰动。
