Substrate-induced conformational changes in yeast 3-phosphoglycerate kinase monitored by fluorescence of single tryptophan probes.
Substrate-induced conformational changes in yeast 3-phosphoglycerate kinase monitored by fluorescence of single tryptophan probes.
复制标题
通过单个色氨酸探针的荧光监测酵母 3-磷酸甘油酸激酶中底物诱导的构象变化。
DOI：
10.1002/pro.5560050616
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Mas,MT
中科院分区：
文献类型：
--
作者：
Cheung,CW;Mas,MT
3‐Phosphoglycerate kinase (PGK) catalyzes the reversible conversion of 3‐phosphoglycerate (3‐PG) and ATP to 1,3‐diphosphoglycerate (1,3‐diPG) and ADP in the presence of magnesium ions. PGK is a single polypeptide chain arranged in two domains, with an active site located in the interdomain cleft. The large distance between the binding sites for 3‐PG and ATP, deduced from the crystallographic structures of the binary complexes, gave rise to the hypothesis that this enzyme undergoes a hinge‐bending domain motion from open to closed conformation during catalysis. However, no direct experimental evidence exists for the “closed” conformation in the presence of both substrates. In this study, several PGK mutants with single tryptophans placed in various locations were used as intrinsic fluorescent probes to examine the extent and delocalization of conformational changes induced by the binding of 3‐PG, 1,3‐diPG, ADP, ATP, and PNP‐AMP (nonhydrolyzable analogue of ATP), and by 3‐PG and PNP‐AMP together. The results showed that only the probes situated in the hinge and in parts of each domain close to the hinge reflect substrate‐induced conformational changes. Binding of substrates to one domain was found to induce spectral perturbation of the probes in the opposite domain, indicating a transmission of conformational changes between the domains. A combination of both substrates generated much larger fluorescence changes than the individual substrates. The binding constants were determined for each substrate using probes situated in different locations.
