George J. Kasperek Connecticut College, New London, 06320 and R. F. Pratt Wesieyan University Middletown. Connecticut 06457
The Fumarase Reaction A nuclear magnetic resonance experiment for biological chemistry students
Over the past 20 years, nuclear magnetic resonance spectroscoov has become one of the most valuable routinelv used physic'8i methods in chemistry. Its usefulness has been amply demonstrated in problems of both a static (for example, structure determination) and a dynamic (reaction kinetics and mechanism) nature. Such is its imuortance that todav it is commonly introduced to students in introductory chemistry courses and varticularlv in organic chemistrv. Advanced chemistry students are, b f course, well acquainted with the technique, both in theory and in practice. For the latter, a numhe; of useful studentkxperimehts have been devised and published (I). Recent years have witnessed the explosive expansion of magnetic resonance techniques into biochemistry where i t is A numher of clear thev have wide and imoortant aoolication. . recent reviews and hooks describe this process (2). In view of these circumstances. we felt that it would be anoronriate to include an experiment using these techniques k o t'he laboratory portion of a biochemistry course. The students involved, mainly juniors and seniors, had been previously introduced to the basic orincioles of nuclear maenetic resonance in organic chemistry and were thus prepared to use and expand their knowledge a t this stage. A search of standard lahoratory manuals and this Journal, however, revealed a scarcitv of suitable experiments of this tvve: hence this work. Many of the applications of n ~ c l e a ~ m a ~ n eresonance tic in biochemistry, such as those involving examination of the spectra of biological macromolecules ;equire sophisticated instrumentation and data processing. Since we, and presumably many other small departments, do not have available ready student access to such equipment, we have devised the following experiment which can be carried out with any small commercial proton magnetic resonance (pmr) spectrometer (in our case, a Perkin-Elmer R-24A) and essentially completed by students in one laboratory period.
fumarate
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t comoaAt eauilibrium, fumarate and malate are ~ r e s e n in rabl~concentrkionsso that both can be &en readily in {mr spectra of the final solution. Essential points concerning the
The experiment examines the fumarase (EC 4.2.1.2) catalyzed conversion of L-malate into fumarate; progress of the reaction is followed bya pmr method. This enzyme and reaction is, of course, one that students will be familiar with from classwork on the citric acid cycle. We feel that it is a useful and instructive exercise for students for a number of reasons 1) It is an example of the application of pmr techniques in hio-
chemistry. 2) It gives students further practical experience in pmr methods
and in pmr spectral assignments. 3) It can be used to illustrate several important stereochemical paints, e.g., the properties of a proehiral center adjacent to a
c h i d center and the stereochemistry (re versus si) of addition to double bonds. 4) It can he used to illustrate, in a way easily appreciated by students. a number of imoortant Dronerties of enzvmes as bioloeical
an reaction equilibria in a way that issimplerandmoreclearto
students than the more common enzyme kinetics eaperiments. Theory The reactions catalyzed by fumarase and initiated by the addition of the enzyme toa solution of L-malate in deuterium oxideare shown below
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PPM . . ...
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Pmr spectra of a solution of 0.2 Msodium malate in D20at pH 7.5: (a) before addition of fumarsse.(b)40 minutesafter addition of 25 units of fumarase,and (c) 24 hr after furnameaddition.
Volume 54, Number 8, August 1977 1 515
structure, function, and mechanism of action of fumarase have been recently reviewed (3). T h e pmr spectrum of L-malate in deuterium oxide (Fig. l(a)) shows a typical ABX splitting pattern where the downfield hydrogen (4.256) on C-2(X) is split to different extents ( J = 4.4 Hz, 9.8 Hz) by the magnetically non-equivalent hydroeens (2.3 6. 2.6 6) on C-3(AB). T h e latter solit each other (J 16.3 Hz) and ake split by the hydrogen a; C-2. On addition of fumarase t o the solution, the malate peaks decrease in intensity and a sharp singlet appears downfield a t 6.5 6. T h e latter, of course, is t h e spectrum of the product fumarate which has two magnetically equivalent hydrogens. An intermediate spectrum is shown in Figure Uh). Eventually, equilibrium is reached when no further increase in the fumarate peak height is observed. Noticeably towards the latter stages of the approach to equilihrium, however, and suhsew e n t t o the establishment of malate/fumarate equilibrium,
