Effects of divalent metal ions on the activity and conformation of native and 3-fluorotyrosine-PvuII endonucleases

被引:28
作者
Dupureur, CM [1 ]
Hallman, LM [1 ]
机构
[1] Texas A&M Univ, Dept Biochem & Biophys, College Stn, TX 77843 USA
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1999年 / 261卷 / 01期
关键词
restriction endonuclease; NMR spectroscopy; 3-fluorotyrosine; divalent metal ions; conformation;
D O I
10.1046/j.1432-1327.1999.00265.x
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 activities of restriction enzymes are important examples of Mg(II)-dependent hydrolysis of DNA. While a number of crystallographic studies of enzyme-DNA complexes have also involved metal ions, there have been no solution studies exploring the relationship between enzyme conformation and metal-ion binding in restriction enzymes. Using PvuII restriction endonuclease as a model system, we have successfully developed biosynthetic fluorination and NMR spectroscopy as a solution probe of restriction-enzyme conformation. The utility of this method is demonstrated with a study of metal-ion binding by PvuII endonuclease. Replacement of 74% (+/- 10%) of the Sr residues in PvuII endonuclease by 3-fluorotyrosine produces an enzyme with Mg(II)-supported specific activity and sequence specificity that is indistinguishable from that of the native enzyme. Mn(II) supports residual activity of both the native and fluorinated enzymes; Ca(nj does not support activity in either enzyme, a result consistent with previous studies. H-1- and F-19-NMR spectroscopic studies reveal that while Mg(II) does not alter the enzyme conformation, the paramagnetic Mn(II) produces both short-range spectral broadening and longer range changes in chemical shift, Most interestingly, Ca(II) binding perturbs a larger number of different resonances than Mn(II). Coupled with earlier mutagenesis studies that place Ca(II) in the active site [Nastri, H.G., Evans, P.D., Walker, I.H. & Riggs, P.D. (1997) J. Biol. Cjem, 272, 25761-25767], these data suggest that the enzyme makes conformational adjustments to accommodate the distinct geometric preferences of Ca(II) and may play a role in the inability of this metal ion to support activity in restriction enzymes.
引用
收藏
页码:261 / 268
页数:8
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