In the Laboratory
A
19F
NMR Study of Enzyme Activity
Keith E. Peterman, Kevin Lentz, and Jeffery Duncan Physical Sciences Department, York College of Pennsylvania, York, PA 17405
Previously, we published in this Journal a laboratory experiment that serves as a model for studying enzyme activity with a basic 60-MHz continuous-wave 1H NMR spectrometer (1). This experiment presented the creativity of NMR biochemistry studies to undergraduates who may not have access to sophisticated FT NMR capabilities. As a new dimension to our previously published 1H NMR experiment, we developed this basic 19F NMR enzyme activity laboratory experiment to demonstrate how 19F NMR can be used in CW or FT NMR biochemical studies and to present the advantages of 19F NMR over 1H NMR for studies of this nature. Although 1H NMR has been widely applied to biochemical studies, 19F NMR is becoming increasingly important as medicinal chemists and biochemists discover the attractiveness of fluorine as a substituent in modifying reactivity (2–4 ). In addition to modifying reactivity, incorporation of fluorine into biological substrates opens a unique broad spectral window for viewing biomolecular structure and dynamics in solution (5). This experiment is pedagogically suited for junior/senior-level courses in instrumental analytical chemistry, biochemistry, molecular biology, or spectroscopy. Discussion
Spectrum 1
Biochemical reactions are normally carried out in aqueous solutions. The solvent consequently becomes a major problem within the 1H NMR spectral window, especially for instruments that lack solvent-suppression capabilities. The broad water peak, along with its spinning sidebands, can obscure the low-amplitude peaks of biomolecular substrates in dilute aqueous solution. However, using a fluorine-tagged substrate avoids the solvent-peak problem entirely. The tagged substrate becomes an excellent analytical reagent for both qualitative and quantitative identification of components in a mixture when analyzed via 19F NMR. Fluorine is a non-stericallydemanding substituent with a van der Waals radius similar to that of hydrogen and it is isoelectronic with the hydroxyl radical. Systematic substitution of F for either H or OH in a molecule can profoundly affect biomolecular activity. The trifluoroacetyl moiety has been the most commonly used group in 19 F NMR biochemical studies (4). N-trifluoroacetylglycine was selected as a model fluorine-tagged substrate because it is commercially available and relatively inexpensive. In addition, glycine is stereochemically inactive, so there is no need to resolve enantiomers. N-trifluoroacetylglycine was found to readily undergo enzyme-catalyzed hydrolysis to produce trifluoroacetic acid and glycine according to the net equation: O
H
H
O
O
C
A
N
C
H
C
OH
H2O
H
O
C
C
H
acylase I CF3
Results of the 19F NMR studies depicting the acylase I (E.C. no. 3.5.1.14)–catalyzed hydrolysis of N-trifluoroacetylglycine are presented in the figure below. This figure illustrates the progress of two different reactions with fixed substrate concentrations but varying amounts of enzyme following a 30 minute incubation period. In Spectrum 1, the hydrolysis reaction has progressed approximately 60%; Spectrum 2 shows the reaction near completion. Note the conversion of the fluorines on the trifluoroacetyl moiety of N-trifluoroacetylglycine (peak A) to the fluorines of trifluoroacetic acid (peak B). The spectra are observed on a 2.0-ppm sweep width.
CF3
C
N
OH + H
B
OH
Spectrum 2
In a typical experiment, a set of 10 samples with fixed substrate concentrations is inoculated with varying concentrations of enzyme. The extent of hydrolysis is determined by comparing the integrated ratios of the two peaks:
% hydrolysis =
integrated area of B × 100 integrated area of A + B
A linear plot is obtained for % hydrolysis versus enzyme concentration. Owing to large relative errors associated with small integrations, it is best to work in a region of 20 to 80% hydrolyzed starting material (1). The slope of the standard plot can be used to calculate unit activity of the enzyme toward this substrate. slope = µ mol substrate per mg enzyme
unit activity =
% hydrolysis 200 µmol substrate × mg enzyme × 0.5 h 100% hydrolysis
Therefore, unit activity = slope × 4. Experimental Details Solution preparation and hydrolysis reactions can be completed in a three-hour laboratory period. Denatured solutions are saved for NMR analysis in a second three-hour laboratory period.
H
JChemEd.chem.wisc.edu • Vol. 75 No. 10 October 1998 • Journal of Chemical Education
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In the Laboratory
Buffer A 13.61-g sample of potassium monobasic phosphate (Fisher) was dissolved in 900 mL of water. The pH was adjusted to 7.00 with a 6.0 M sodium hydroxide solution. The buffer solution was adjusted to a final volume of 1000 mL with water. Substrate Solution A stock solution of substrate was prepared by dissolving 500 mg of N-trifluoroacetylglycine (Sigma) per 20 mL of buffer and adjusting the pH to 7.00 with sodium hydroxide. NOTE: It is important to readjust the substrate solution to a pH of 7.00 or the enzyme will be inactive! Enzyme A stock solution of enzyme was prepared by dissolving 24 mg of acylase I (Sigma no. A-7264) per 12 mL of buffer. One-milliliter samples of stock solutions were diluted with buffer to produce enzyme solutions with standard concentrations ranging from 0.040 to 0.200 mg of enzyme per milliliter of buffer. Hydrolysis Reaction Each vial containing 2 mL of stock substrate solution (200 µmol) was inoculated with 1 mL of enzyme solution and incubated in a constant-temperature water bath at 35 °C for 30 minutes. After the incubation period, the enzyme was denatured by placing the vial in a boiling water bath for 10 minutes. NMR Spectrometric Analysis Spectra for each sample were analyzed on a Varian EM360-L NMR spectrometer equipped with a 19F probe. The spectra were observed over a 2.0 ppm sweep width from δ -73 to δ -75 relative to an external CCl3F reference. The chemical shift difference between peaks A and B is 0.25 ppm (15 Hz on a 60-MHz NMR). Percent hydrolysis for each sample was evaluated from integrated ratios of the two observed peaks.
1284
Conclusion This experiment demonstrates how 19F NMR can serve as an excellent analytical tool for biochemical studies. Students can clearly see how incorporation of fluorine into a biological substrate opens a wide spectral window with minimal background interference (5). The 19F nucleus can be compared to the 1H nucleus showing like nuclear spins (I = 1/2), similar sensitivities (19F signal is 83% the strength of the 1H signal), similar natural abundances (≈100%), and large disparity in standard chemical shifts (0 to 10 ppm for proton versus +50 to ᎑250 ppm for fluorine) (5, 6 ). Further, the experiment can be used to introduce the chemical attractiveness and utility of fluorine as a non-sterically-demanding substituent that shows pronounced electronic effects in biologically active molecules (2–4). The methods employed in this experiment can be extended to other enzyme–fluorinated substrate systems and can be analyzed on any CW or FT NMR equipped with a 19F probe. Literature Cited 1. Peterman, K. E.; Labenski, J. P.; Hamberger, T. L.; Pinkowski, C.; Raub M. J. Chem. Educ. 1989, 66, 875–876 . 2. Welch, J. T.; Eswarakrishnan, S. Fluorine in Bioorganic Chemistry; Wiley: New York, 1991. 3. Welch, J. T. In Selective Fluorination in Organic and Bioorganic Chemistry; Welch, J. T., Ed.; American Chemical Society: Washington, DC, 1990; Chapter 1. 4. Ojima, I.; McCarthy, J. R.; Welch, J. T. Biomedical Frontiers of Fluorine Chemistry; American Chemical Society: Washington, DC, 1996. 5. Everett, T. S. In Chemistry of Organic Fluorine Compounds II; Hudlicky, M.; Pavlath, A. E., Eds.; American Chemical Society: Washington, DC, 1995; pp 1037–1086. 6. Mooney, E. F. An Introduction to 19F NMR Spectroscopy; Heyden: London, 1970.
Journal of Chemical Education • Vol. 75 No. 10 October 1998 • JChemEd.chem.wisc.edu
