An NMR Study of the Stereochemistry of the Fumarase-Catalyzed Hydration of Fumaric Acid Julie A. Olsen and Robert J. Olsen Wabash College, Crawfordsville, IN 47933 One of the earliest biochemical applications of 'H-NMR spectrosocpy was a study that determined the stereochemistry of the fumarase-catalyzed hydration of fumaric acid, eq 1(14).We have used a 'H-NMR study of this reaction as an experiment in our organic chemistry course for chemistry majors and in an advanced course in hioanalytical chemistry for nonmajors. The conditions of thereaction aresuch that i t
can he followed easily, and the stereochemistry of the product determined using 60-MHz 'H-NMR.
t~~~
-0zc H
1
COz-
H OH
(I)
The reaction involves the conversion of fumarate to malate, catalyzed by furmarase (fumarate hydratase, E.C. 4.2.1.2 from chicken heart). The addition of water to the double hond is stereos~ecific.This can he observed by performing the reaction i n ~ 2 0which , results in the prod&on of two chiral cencers. The vroduct will be one of two possible diastereomers, each ha& a different coupling constant for its Droton sianals. In the substrate the two hydrogens located o n i h e olefinic carbons appear as a singlet~at6.7 ppm. The only other observable peak, a t 5.0ppm, comes from the HOD formed when the phosphate buffer is prepared in Dz0. The addition of DzO to the double hond can in principle he either syn or anti giving the diastereomers shown in eq 2. With syn addition the product would be 2S,3S-malate with the two nrotons in a eauche confirmration. With anti addition the product woul&be 2 ~ , 3 ~ - m i l a tand e , the protons would he in an anti confieuration. The K a r ~ l u eauation s predicts a coupling constant of 1.8 Hz for thesyn product (gauche configuration, 60° HCCH dihedral angle) and 9.2 Hz for the anti product (anti configuration, 180' HCCH dihedral angle) (78.
.~
~~~
PPM 7
6
5
4
3
2
Figure 1. Initial (a) ard final (b, 72 h) NMR spectra of the fumarase catalyzed hydration of fumarate in D20.
436
Journal of Chemical Education
gy conformationsof the two diastereomers of the malate dianion deuterated at C-3. Using the dihedral angles between the C-2 and C-3 orotons for these lowest enerw conformationsand the Karplus'equation, calculate the proton coupling constants for each diastereomer. We have used PCMODEL1 and found that the hydrogen bonded conformation shown in Figure 2 is the most stable with H-GC-H dihedral angles of 17B0and 64'. 4. Consider the effect of the deuterium on the proton NMR spectrum.
Figure 2. dianion.
PGMOOEL-generated minlmum energy conformallon ol the malate
The enzymatic product shows a doublet centered a t 4.6 ppm for the proton on C-2 and a doublet of triplets a t 2.7 ppm for the proton on C-3. The vicinal coupling constant was found to be 9.4 Hz consistent with anti addition. The additional splitting of 1.9 Hz, seen in the proton signal from C-3, is due to geminal proton-deuterium coupling. A triplet is produced because of the I = 1value for deutrium. (See Fie. 1.) The product/suhstrate ratio can be determined by integratiooof the 'H-NMR spectra. The reaction comes toequilihrium after several days at room temperature with a productlsubstrate ratio of -6. Afrer the addition of more substrate, malate production resumes, showing that enzyme ,., values tabulated activity is still present. Using Kmand V by Nigh, (9) a K., of 4.4 for the reaction in H20was calculated. The reverse reaction, the conversion of malate to fumarate, is much slower than the forward reaction (the forward and reverse activation energies are 10 kcallmol and 15 kcall mol, respectively) and is not observed under our experimental conditions (10,II). Depending on the course in which the experiment is used, the students are asked to do all or part of the following activities before beginning the laboratory work. molecular models to examine the oroducts of svn and anti addition, prrdicr the mop1 stahle ronformatmns, and translate the ~ t r u c t u r rinto ~ Newman yrujrctions. 2. Estimate the dihedral angles between the twgl protons in each structure, and use the Karplus equation to compute coupling constants. 3. Use a molecular mechanics program to calculate the lowest ener1. Use ~
~
~~~~
~
-
' Obtained from Serena Software, Box
47402.
3076, Bloomingion, IN
We have also used the laboratory as a review of buffer calculations; the students were asked t o calculate, prepare, and check the p H of the phosphate buffer used. They are also asked to consider the extent of ionization of the carboxylate and hydroxyl groups of the substrate and product a t that pH. An experiment exploring the kinetics of this reaction, published by W. G. Nigh in this Journal (9) would serve as an excellent companion experiment.
Solution Preparation Potassium phosphate buffer, 10mL, 0.01 M, pH 7.3, is prepared in D20.One-millilitersamples of 0.6 M disodium fumsrate and L-(-)malie acid are prepared using the phosphate buffer. Reference Spectra Spectraof rhesuhrtrateand product (undeurerated)are run one Varian T-60 and peak assignments are made. TMS is used as an external standard. Enzyme Reaction Fifteen units uf commercial chicken heart fumaraae (obtained frum Sigma Chemical C O Ii~ added to the fumarate solution (0.6.M in 1 ml. of 0.01M,pH 7.3 -phosphate buffer in DO) to initiate the reaction. The react& is run at room temperature. Spectra run after 30 min will show small product peaks, and sufficient mslate is oroduced within a few hours to make the stereochemical assiznmQn1.Srwrnl days are required lo reach equilihrrum. Integratic~ns uf the fumnrate singlet end malate dnuhlet of triplets allows the reaction to be fdlmed quantitatively. ~
.~~~~
Acknowledgment Financial support of the Haines Research Fund a t Wabash College is gratefully acknowledged. Literature Clted 1. Jsrdatzky, 0.: Robert*, G.C.K. NMR in Moleeulor Biology: Academic: New Yort, 1981;pp 420-421. 2. Farrar,T.C.;Gutoa9ky,H.S.;Alberfy,R.A.:Miller,W.G. J.Am.Chem.Soc. 1957.79, 3978-3980. 3. Alberty, R. A.; Bender. P. J. Am. Chem. Sac. 1959,81, E42-546.
4. Krasna.A. I. J.Biol. Cham. 1958,233.1010-1013. 5. Gswran. 0.;Fondy. T. P. J.Am. Chem. Soc. 1959.81.6333-6334. 6. Anet,F.A.L.J.Am.Chem. Sor. 1966,82.99&995. 7. Kerp1us.M. J . Chem.Phya. 1959.30, 11-15. 8. Scovell, W. M J ChamEduc. 1989.66,111-117. 9. Nigh, W. G.J. Cham.Educ. 19?6,63.668-669. 10. Masney. V. Biochem. J . 1953.53, 72-79. 11. Diron, M.; Webb. E. C . Enzymss, 2nd ad.: Academic: Nea. York,
Volume 66
Number 5
1964:p 164.
May 1991
437
