Pineda T, Kwon O S, Serpersu E H, Churchich J E
Department of Biochemistry, University of Tennessee, Knoxville 37996-8040.
Eur J Biochem. 1993 Mar 15;212(3):719-26. doi: 10.1111/j.1432-1033.1993.tb17710.x.
Yeast 3-phosphoglycerate kinase is inactivated by incubation with pyridoxal 5'-diphospho-5'-adenosine (AdoP2Pxy) [Tamura, J. K., Rakov, R. D. & Gross, R. L. (1986) J. Biol. Chem. 261, 4126-4133). Incorporation of 1 mol affinity label/mol enzyme was sufficient for complete inactivation of 3-phosphoglycerate kinase. The substrate ATP affords substantial protection against inactivation. Partial protection is afforded by the substrate glycerate 3-phosphate. When AdoP2Pxy-modified phosphoglycerate kinase was reduced with [3H]NaBH4 and subjected to trypsin hydrolysis, only one radioactive peptide was isolated by reverse-phase high-performance liquid chromatography. The amino acid composition and sequence analysis of the purified radioactive peptide revealed that it spans residues 379-403 of the enzyme and Lys385 specifically reacted with the affinity label. This peptide represents the hinge region between the two domains of the protein, where the active site is also located. The fluorescence intensity of enzyme-bound AdoP2Pxy is enhanced when glycerate 3-phosphate is added, suggesting exposure of the fluorescent probe to a more hydrophobic environment. Another fluorescent analog, anthraniloyl-dATP (ant-dATP), which carries the fluorescent reporter group on the ribose ring, binds to the enzyme at two distinct sites with Kd values of 6 +/- 2 microM and 25 +/- 3 microM, as determined by steady-state anisotropy measurements. Bound ant-dATP was displaced from the enzyme by glycerate 3-phosphate and ATP, as monitored by the fluorescence anisotropy. These results suggest that both fluorescent ATP analogs bind to the active site, which is at the hinge region of the enzyme. Model-building studies showed that when AdoP2Pxy is built into the open form of the enzyme, as described in X-ray studies, the pyridoxyl group of AdoP2Pxy cannot reach Lys385 for Schiff-base formation. Labeled Lys385 is on a beta-turn immediately following helix XII, which was suggested to interact with the nucleotide and become ordered at the active site of 3-phosphoglycerate kinase [Watson, H. C., Walker, N. P. C., Shaw, P. J., Bryant, T. N., Wendell, P. L., Fothergill, L. A., Perkins, R. E., Conroy, S. C., Dobson, M. J., Tuite, M. F., Kinesman, A. J. & Kinesman, S. M. (1982) EMBO J. 1, 1635-1640]. The results presented here suggest that binding of substrates cause significant structural changes in the enzyme.
酵母3 - 磷酸甘油酸激酶与5'-二磷酸 - 5'-腺苷吡哆醛（AdoP2Pxy）一起温育会被灭活[Tamura, J. K., Rakov, R. D. & Gross, R. L. (1986) J. Biol. Chem. 261, 4126 - 4133]。每摩尔酶掺入1摩尔亲和标记足以使3 - 磷酸甘油酸激酶完全失活。底物ATP能提供显著的抗失活保护。底物3 - 磷酸甘油酸提供部分保护。当用[³H]NaBH₄还原AdoP2Pxy修饰的磷酸甘油酸激酶并进行胰蛋白酶水解时，通过反相高效液相色谱仅分离出一个放射性肽段。纯化的放射性肽段的氨基酸组成和序列分析表明，它跨越酶的379 - 403位残基，且Lys385与亲和标记特异性反应。该肽段代表蛋白质两个结构域之间的铰链区，活性位点也位于此处。添加3 - 磷酸甘油酸时，酶结合的AdoP2Pxy的荧光强度增强，表明荧光探针暴露于更疏水的环境中。另一种荧光类似物，核糖环上带有荧光报告基团的邻氨基苯甲酰 - dATP（ant - dATP），通过稳态各向异性测量确定，它以6±2 μM和25±3 μM的解离常数（Kd值）在两个不同位点与酶结合。如通过荧光各向异性监测的那样，结合的ant - dATP被3 - 磷酸甘油酸和ATP从酶上置换下来。这些结果表明两种荧光ATP类似物都结合到酶的活性位点，该活性位点位于酶的铰链区。模型构建研究表明，如X射线研究中所述，当将AdoP2Pxy构建到酶的开放形式中时，AdoP2Pxy的吡啶基团无法到达Lys385以形成席夫碱。标记的Lys385位于紧接螺旋XII后的一个β - 转角上，有人提出该螺旋与核苷酸相互作用并在3 - 磷酸甘油酸激酶的活性位点处变得有序[Watson, H. C., Walker, N. P. C., Shaw, P. J., Bryant, T. N., Wendell, P. L., Fothergill, L. A., Perkins, R. E., Conroy, S. C., Dobson, M. J., Tuite, M. F., Kinesman, A. J. & Kinesman, S. M. (1982) EMBO J. 1, 1635 - 1640]。此处呈现的结果表明底物的结合会导致酶发生显著的结构变化。
