Synthesis, kinetics, and structural studies of a photolabile caged isocitrate: a catalytic trigger for isocitrate dehydrogenase.
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 binding site but no observable bound isocitrate, whereas after photolysis the enzyme is complexed to liberated isocitrate and Mg2+ with binding interactions identical to those of previously determined substrate complexes. Single-crystal spectroscopy of the crystals after flash photolysis in the presence of substrates shows production of bound enzyme-substrate complexes and reduced nicotinamide adenine dinucleotide phosphate induced by the photolytic event.
Brubaker
M J
MJ
Center for Advanced Materials, Lawrence Berkeley Laboratory, University of California, Berkeley, 94720, USA.
Dyer
D H
DH
Stoddard
B
B
Koshland
D E
DE
D000595:Amino Acid Sequence; D002384:Catalysis; D018360:Crystallography, X-Ray; D005583:Fourier Analysis; D007202:Indicators and Reagents; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D007523:Isocitrates / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010782:Photolysis; D011487:Protein Conformation; D011789:Quantum Theory; D012996:Solutions; D013997:Time Factors
D007202:Indicators and Reagents; D007523:Isocitrates; D012996:Solutions; C098539:caged isocitrate; D007521:Isocitrate Dehydrogenase; D008274:Magnesium