An NMR Study of Enzyme Activity Keith E. Peterman, James P. Labenski, Terry L. Hamberger, Charlene Pinkowski, and Michael Raub York College of Pennsylvania, Country Club Rd., York, PA 17403 During recent years, NMR has increasingly heen applied to biochemical studies. Since most of these studies require sophisticated Fourier transform NMR capabilities, undergraduate students often miss the excitement and creativity associated with these applications for lack of access to the required tools. We have developed this lahoratory experiment as a model for studying enzyme activity with a basic tra are analyzed 60-MHz continuous-wave following NMR the asymmetric spectrometer. hydrolysis NMR of specNacetyl-L-methionine with acylaseI (E.C. no. 3.5.1.14). Multiple substrates are also employed in a competitive hydrolysis enzyme reaction by acylase I. Dlscusslon N-acyl derivatives of methionine are the most susceptible of all substrates toward hydrolysis in enzymic reactions catalyzed by acylase I.' One such substrate, N-acetyl-L-methionine, serves as a good model for demonstrating the use of NMR in enzyme studies. The N-acetyl-L-methionine is hydrolyzed to produce acetic acid and ~ m e t h i o n i n eaccording to the net equation: O
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Results of the NMR studies depicting the acylase I catalyzed hydrolysis are presented in Figure 1. This figure illustrates the progress of three different reactions with fixed substrate concentrations but varying amounts of enzyme following a 30-min incubation period. In spectrum 1of Figure 1,the hydrolysis reaction has not progressed appreciably. Spectrum 2 represents a reaction approximately 50% complete, and spectrum 3 characterizes . .~ complete hydrolysis. The 'pectra are observed On a 0.5-p~msweep width 61.5 to 2.0. In these spectra, the acetyl protons (B) and SCH3 protons (A) of N-acetyl-L-methionine are observed to decrease in intensity with concomitant appearance of peaks for
the acetyl protons of acetic acid (D) and SCH3 protons of methionine (C). For the purpose of this study, a set of samples with fixed substrate concentrations are inoculated with varying concentrations of enzyme. The extent of hydrolysis is determined by comparing the integrated area of the acetyl protons of acetic acid (D) versus the sum of peaks B and D. (integrated area of D) x 100 %hydrolysis= X = (integrated area of B D)
+
A linear plot is obtained for X versus enzyme concentration. Due to a large relative error associated with small integration curves, it is best to work in a region of 20% to 80% hydrolyzed. The NMR results compare favorably with those obtained via a standard UV analysis on the same ample.^ In a competitive reaction, a two-substrate mixture containing N-acetyl-L-methionine and N-acetyl-(DL)-o-fluorophenylalanine is inoculated with acylase I, incubated, and heat denatured after 30 min. Representative samples of an unhydrolyzed mixture (spectrum 1). a partially completed reaction (spectrum 2), and a completed reaction (spectrum 3) are presented in Figure 2. As evidenced in the single substrate study, peaks A and B decrease in intensity with concomitant appearance of peaks C and D.
~
Presented in part at the 15th Middle Atlantic Regional Meeting of the American Chemical Society and the 58th Annual meeting of the pennsylvania~~~d~~~ of sciences. Fontes, W. S.: Lee. M. J. Biol. Chem. 1952. 201. 857. Sigma. Enzymatic Assay of Acylase L 1976
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Volume 66 Number 10 October 1989
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Figwe 3. NMR spectra for and aft% acidilication.
Figwe 2. NMR s p a n rspreDeminghydmMisof mixed substrates Nacetyi-Lmeihionine and Nacetyymt~fiuwophenylalanine.
The chemical shift of peak D, representing the acetyl protons of acetic acid, coincides with peak E representing the N-acetyl protons of the N-acetyl-(DL)-o-fluorophenylalanine. However, since the mole ratio of N-acetyl-L-methiis 1to 1, onine versus N-acetyl-(DL)-o-fluorophenylalanine the percent hydrolysis of N-acetyl-L-methionine is computed from the areas of peaks B, D, and E. (integratedarea of B) X 100 %hydrolysis= X = 100 (integrated areas of B + D + E)/2 Although the overlapof peaks D and E negate direct calculation of percent hvdrolysis of N-acetyl-(DL)-o-fluorophenylalanine,-the t ~ o - ~ e a kcan * be resolved by adding a small amount of HCI t o the NMR tube containing the mixed substrates. -~ A downfield shift of the acetvl Drotons of acetic acid (D, Fig. 3) is induced since acidification shifts the acetic acid + acetate ion equilibrium toward the molecular acid such that the time-averaged environment observed on the NMR spectrum represents a more deshielded species. Resolution of all three peaks B, D, and E permits direct comparative inteeration of the N-acetvl protons for each suhstrateversus acetic acid. The enzGe activity toward N-acetyl-Lmethionine in the mixed substrate system remains the same as is evidenced in a coincident single substrate hydrolysis reaction. The N-acetyl-(DL)-o-fluorophenylalaninein the mixed substrates system neither hydrolyzes nor inhibits the competing reaction. ~~~
Buffer
A 13.61-g sample of potassium monobasic phosphate (Fisher) was dissolved in 900mL water. The pH was adjusted to 7.00 withsodium
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Journal of Chemical Education
mmplated reactim wiU7 mixed substrates at pH 7
hydroxide. The solution was adjusted to a final volume of 1000 mL with HzO. Substrate Solutions
Stock solutions containing 1M) ,mol substrate per milliliter of buffer were prepared hy dissolving the appropriate substrate in phosphate buffer, 382 mg N-acetyl-L-methionine(Sigma) per 20 mL buffer or 225 mg N-acetyl-(DL)-o-fluorophenylalanine(Sigma) per 10mL buffer, and adjusting the pH to 7.00 with sodium hydroxide. A stock solution was prepared by dissolving 10.0 mg acylase I (Sigma no. A-7264) per 20 mL buffer. One-milliliter samples of the stock solutions were diluted with buffer to oroduce enzvme salut& with concentrations ranging from 0.01 to 0.10 mg enzyme per milliliter of buffer.
Hydrolysis Reactions
Each vial containing the substrate (or substrates) to be hydrolyzed (2M) ,nnol/2 mL buffer) was inoculated with 1 mL of the enzyme solution and incubated at 25 O C for 30 min. Following the incubation period, the enzyme was beat denatured by placing the vial in a boiling water bath for 10 min. NMR Spectrometric Assay Spectra for each sample were recorded on a Varian EM360-L NMR spectrometer.The spectra was obsemed over a OB-ppm sweep width from 61.5 to 62.0 relative to an external TMS reference. Percent hydrolysis for each sample was evaluated from integrated ratios of select peaks.
Conclusion This experiment demonstrates how NMR can serve as an analytical tool for the study of enzyme activity. Students can see the capability of NMR spectroscopy in hiochernical studies. The methods employed in this experiment can be extended to other enzyme-substrate systems.
