C-13 NMR-STUDIES OF THE ENZYME PRODUCT COMPLEX OF BACILLUS-SUBTILIS CHORISMATE MUTASE

被引:26
作者
RAJAGOPALAN, JS [1 ]
TAYLOR, KM [1 ]
JAFFE, EK [1 ]
机构
[1] FOX CHASE CANC CTR,INST CANC RES,7701 BURHOLME AVE,PHILADELPHIA,PA 19111
关键词
D O I
10.1021/bi00066a017
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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. C-13 NMR was used to study the structure of [U-C-13]prephenate bound at the active site of B. subtilis chorismate mutase. All the enzyme-bound C-13 NMR resonances of [U-C-13]prephenate were assigned, and where possible, 1J(C,C)s were quantified; [1,3,5,8-C-13]prephenate and [2,6,9-C-13]prephenate, prepared respectively from [1,3,5,8-C-13]-chorismate and [2,6,9-C-13]chorismate, aided the C-13 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. C-13 NMR studies of model compounds at various pH values and in various solvents suggest that the observed C-13 chemical shift changes of enzyme-bound prephenate cannot be rationalized solely on the basis of changes in the pK(a)s of the carboxylic acid groups or hydrophobic solvation at the active site. With regard to the chemical mechanism of the chorismate mutase-catalyzed reaction, these NMR studies do not provide any evidence for a dissociative mechanism which involves discrete intermediates.
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页码:3965 / 3972
页数:8
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