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. 1974 Feb;71(2):469–473. doi: 10.1073/pnas.71.2.469

Fluorotyrosine Alkaline Phosphatase from Escherichia coli: Preparation, Properties, and Fluorine-19 Nuclear Magnetic Resonance Spectrum

Brian D Sykes *, Harold I Weingarten †, Milton J Schlesinger ‡
PMCID: PMC388028  PMID: 4592693

Abstract

Alkaline phosphatase (EC 3.1.3.1) containing m-flurotyrosine has been prepared from E. coli grown in the presence of m-flurotyrosine. The kinetic properties of the m-fluorotyrosine enzyme measured with p-nitrophenylphosphate at pH 8.0 and dinitrophenylphosphate at pH 5.5 are essentially the same as those of normal alkaline phosphatase. However, the ability of the m-fluorotyrosine protein to refold active enzyme after acid denaturation, while unchanged at pH 5.8, was markedly decreased at pH 7.6. This result implies that the tyrosines must be in their protonated form for the protein to refold, reassociate, and take on zinc. The 19F nuclear magnetic resonance spectrum of m-fluorotyrosine alkaline phosphatase contains resolved resonances corresponding to different chemical environments for each m-fluorotyrosine in the folded protein. This demonstrates that 19F nuclear magnetic resonance spectroscopy of enzymes specifically labeled with 19F, even with enzymes as large as alkaline phosphatase (molecular weight, 86,000), will provide a very valuable probe for conformational changes in proteins.

Keywords: kinetics, renaturation, ultraviolet spectra, conformational changes

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Selected References

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