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Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy
Published on: June 27, 2014
Infrared studies reveal unique vibrations associated with the PGK-ATP-3-PG ternary complex
Ellen M White1, Amanda R Holland, Gina MacDonald
1Department of Chemistry, James Madison University, Harrisonburg, Virginia 22807, USA.
Biochemistry
|December 15, 2007
Summary
Difference infrared spectroscopy reveals unique conformational changes in phosphoglycerate kinase (PGK) when bound to ATP and 3-phosphoglycerate (3-PG). These changes involve the hinge region and specific amino acid side chains, offering insights into enzyme catalysis.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Phosphoglycerate kinase (PGK) is crucial for energy metabolism, catalyzing a reversible reaction between ATP and 3-phosphoglycerate (3-PG).
- This reaction is believed to involve a hinge-bending motion, bringing substrate-binding domains together.
- Understanding these conformational dynamics is key to elucidating PGK's catalytic mechanism.
Purpose of the Study:
- To investigate the unique conformational changes in PGK during catalysis using difference infrared spectroscopy.
- To identify specific structural alterations associated with the formation of the PGK-ATP-3-PG ternary complex.
- To correlate spectroscopic findings with enzyme activity assays.
Main Methods:
- Utilized difference infrared spectroscopy to monitor conformational changes in PGK.
- Employed caged nucleotides (caged-ATP and caged-ADP) to initiate controlled substrate binding.
- Performed complementary enzyme activity assays with caged nucleotides.
Main Results:
- Difference FTIR spectra revealed distinct bands associated with the PGK-ATP-3-PG complex, indicating structural changes in the hinge region.
- Specific amino acid side chains (Arg, Asn, His, Lys, Asp, Glu) were identified as being perturbed in the active ternary complex.
- Activity assays showed that caged-ADP inhibits PGK, while caged-ATP does not, suggesting differential nucleotide interactions.
Conclusions:
- The study provides spectroscopic evidence for conformational changes, including alterations in alpha-helix and beta-structures, unique to the active PGK ternary complex.
- These findings support the hinge-bending model and highlight the role of specific amino acid residues in PGK's catalytic mechanism.
- Difference infrared spectroscopy is a valuable tool for dissecting enzyme conformational dynamics during substrate binding and catalysis.
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