13C NMR studies of the enzyme-product complex of Bacillus subtilis chorismate mutase.
The chorismate mutase reaction is a rare enzyme-catalyzed 3,3-sigmatropic rearrangement of chorismate to prephenate. Bacillus subtilis chorismate mutase was overproduced and purified from Escherichia coli XL1-Blue (pBSCM2) using a modification of the procedure of Gray et al. (Gray, J. V., Grolinelli-Pimpaneau, B., & Knowles, J. R. (1990) Biochemistry 29, 376-383); the modification leads to minimal contaminating prephenate dehydratase activity (< 0.001%). The native molecular mass of B. subtilis chorismate mutase was determined by gel filtration to be approximately 44 kDa, indicative of a homotrimer of the 14.5-kDa subunits as determined by electrospray mass spectrometry. 13C NMR was used to study the structure of [U-13C]prephenate bound at the active site of B. subtilis chorismate mutase. All the enzyme-bound 13C NMR resonances of [U-13C]prephenate were assigned, and where possible, 1JC,Cs were quantified; [1,3,5,8-13C]prephenate and [2,6,9-13C]prephenate, prepared respectively from [1,3,5,8-13C]chorismate and [2,6,9-13C]chorismate, aided the 13C NMR resonance assignments. Enzyme-bound prephenate exhibits remarkably different chemical shifts relative to free prephenate; the chemical shift changes range from -6.6 ppm for the C6 resonance to 5.6 ppm for the C5 resonance, suggesting a strong perturbation of the C5-C6 bond. 13C NMR studies of model compounds at various pH values and in various solvents suggest that the observed 13C chemical shift changes of enzyme-bound prephenate cannot be rationalized solely on the basis of changes in the pKas of the carboxylic acid groups or hydrophobic solvation at the active site.(ABSTRACT TRUNCATED AT 250 WORDS)
Rajagopalan
J S
JS
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Taylor
K M
KM
Jaffe
E K
EK
D001412:Bacillus subtilis / Q000201:enzymology*; D002247:Carbon Isotopes; D002826:Chorismate Mutase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002827:Chorismic Acid / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003509:Cyclohexanecarboxylic Acids / Q000378:metabolism; D053138:Cyclohexenes; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D002247:Carbon Isotopes; D003509:Cyclohexanecarboxylic Acids; D053138:Cyclohexenes; D011994:Recombinant Proteins; D002826:Chorismate Mutase; D002827:Chorismic Acid; C005550:prephenic acid