Substrate-induced conformational changes in yeast 3-phosphoglycerate kinase monitored by fluorescence of single tryptophan probes.
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 location 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.
Cheung
C W
CW
Division of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Mas
M T
MT
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D004163:Diphosphoglyceric Acids / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005988:Glyceric Acids / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D016296:Mutagenesis / Q000235:genetics; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D017186:Titrimetry; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
D004163:Diphosphoglyceric Acids; D005456:Fluorescent Dyes; D005988:Glyceric Acids; C015891:glycerate 1,3-biphosphate; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; C005156:3-phosphoglycerate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D010735:Phosphoglycerate Kinase; D008274:Magnesium