Synthesis, kinetics, and structural studies of a photolabile caged isocitrate: a catalytic trigger for isocitrate dehydrogenase

Biochemistry
M BrubakerD E Koshland

Abstract

A biologically inactive photolabile derivative of isocitrate has been synthesized and characterized. The caged isocitrate is photolyzed to isocitrate with a rate constant of 234 s-1, a half-life of 3 ms, and a quantum yield of 0.3 at pH = 6.4. Caged isocitrate (1-(2-nitrophenyl)ethyl 1-hydroxy-1,2-dicarboxy-3- propanecarboxylate) was synthesized in a straightforward synthetic manner starting with racemic isocitric acid lactone. Laser pulse photolysis at a wavelength of 355 nm was used to determine the rate of photolysis and the quantum yield and to quantify the amount of energy needed for quantitative conversion of the caged isocitrate to free isocitrate. Enzymatic conversion of the liberated isocitrate to alpha-ketoglutarate was achieved in solution as well as in wild-type and mutant isocitrate dehydrogenase (IDH) protein crystals. The X-ray crystal structures of wild-type IDH soaked with photolabile caged isocitrate and Mg2+ and void of nicotinamide adenine dinucleotide phosphate were solved at 2.5 A resolution before and after photolysis and compared by difference mapping against previously determined enzyme structures. Prior to photolysis the enzyme active site contains a low occupancy of bound free Mg2+ in the metal bindin...Continue Reading

References

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Citations

Aug 16, 2012·Biochemistry·Susana GonçalvesPedro M Matias
Oct 23, 1998·Nature Structural Biology·B L StoddardD E Koshland
Jun 17, 1998·Nucleic Acids Research·S G Chaulk, A M MacMillan
Mar 27, 2012·Bioorganic & Medicinal Chemistry Letters·Chandan MaitraniRobert S Phillips
Jun 1, 2001·Methods : a Companion to Methods in Enzymology·B L Stoddard
May 24, 2000·Organic Letters·P D KehayovaA Jain
Mar 15, 2003·Science·Sushmita D LahiriKaren N Allen

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