Using High Field NMR to Determine Dehydrogenase Stereospecificity with Respect to NADH An Undergraduate Biochemistry Lab Sara 6. Mostad and Atthur Glasfeld Reed College, Portland, OR 97202 As catalysts, enzymes are notable for their high level of substrate specificity and stereoselectivity. Enzymes typically catalyze the transformation of a narrow range of substrates and furthermore handle only one stereoisomer. A more subtle form of this stereoselectivity can be found in some enzymes' ability to select between two atom groups related by a mirror plane of symmetry in the substrate. Enzymatic selectivity for particular enantiotopic or diastereotopic atoms or fundional gmups has only been recognized for only about 40 years (1).It is frequently challenging to identify this level of stereospecificity in enzymatic catalysis, but the results can be pivotal in uncovering mechanistic information. The importance of stereospecificity in biochemistry has led to the development of a number of undergraduate laboratory experiments (2, 3). The experiment described here investigates the stereoselectivityof dehydrogenase-catalyzed hydrogen transfer to NAD(P)+,using high field NMR spectroscopy. The procedure offers junior and senior biochemistry students, who mav have ex~erienceusine low field instrumerits, an oppokunily to'discover the additional information that is available from high field spectra.
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Background In the early 1950's, Vemesland and Westheimer performed pioneering work that showed dehydmgenases have specificity for one or the other of two diastereotopic hydmgens, designatedpro-R andpm-S, a t C4 of the dihydmniwtinamide ring of NADH (Fig. 1)(4). In the last 40 years, determinations of stereospecificities for well over 150 dehydrogenases have been performed, and each one has absolute stereospecificity (5). The wealth of data has prompted several investigators to search for trends between the substrate and stereospecificity with respect to NAD(P)H and has resulted recently in a vigorous debate (6, 7).
Figure 2. The folded conformation of HADH. The ring current of the adenine ring selectively shields the "6"hydrogen. Note that the distoltion of the dihydronicotinamide ring puts the "A" hydrogen out of the plane of the ring, reducing coupling to the proton on C5 of the ring. Kaplan and co-workers first showed that 220 MHz NMR spectra can be used to identifv the ~ r o - R and o m 3 hvdw gens (8). The two protons have disthct chemic'alshifts, due to a folded conformation of NADH in solution. The pro-S hydrogen faces the adenine ring of NADH and is shielded by the ring current, leading to a slightly upfield chemical shiR relative to the pro-R hydrogen (Fig. 2). This conformation is furthermore believed to lead to a puckering of the dihydmnicotinamide ring, placing thepro-R hydrogen in an axial position. The two C4 protons also may be distinguished by their coupling constants to the proton on C5. A Karplus correlation can be used to show that thepro-S hydrogen, which shows greater splitting due to the C5 pmton, is more closely aligned with it. By using deuterated hydroxyl substrates, a student may readily prepare one of the two C4 deuterated diastereomers of NADH, which will reveal the stereospecifity of hydrogen transfer. The following experimental procedure describes the preparation of deuterated NADH for study by NMR. Students will reduce NADiwith a deuterated substrate in the presence of a dehydrogenase and purify the product 4I2H1NADH from substrate prior to characterization by NMR. The experiment offers opportunities for students to perform enzyme assays and column chromatography, in addition to gaining experience with NMR spectroscopy. Exnerimental Procedures Materials
Figure 1. The structure of NADH. The 5' hydroxyl of the ribose forms a phosphate ester linkage with the p phospate of adenosine diphosphate (ADP). Tht HRhydrogen is the p r o 4 hydrogen and prh5 hydrogen is labeled Hs. 504
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ld-glucose, ds-2-propanol, D20and D20with 0.75%TSP were purchased from Aldrich Chemical Co. NAD+,NADH, DEAE cellulose, glucose dehydrogenase from Baeillr~sand alcohol dehydrogenase from horse liver were purchased
the conversion of 1 pmole of NAD+toNADH per minute. The extinction coeffikent for NADH is 6220 M-'em-'. Stereospeclflclty of Alcohol Dehydrogenase Eight units of alcohol dehydrogenase were added to a reaction mixture containing 2.0 mL of 7 mM NAD' (10 mg) and 3.2 M d.-2-orooanol (500 uL added volume) in sodipm phosphate buffer (50 mM, pH 8.5) at 25 OC. Reaction progress was followed by removing 10-pL aliquots from the reaction mixture, diluting them to 1mL with buffer and checking the absorbance at 340 nm. When the reaction was judged to be greater than 75% comvlete (after about 15-20 mini, the reaction mixture was transferred to a small roundbottomed flask and evaporated under reduced pressure at 55 'C. The material was redissolved in 2 mL of D20, the solution evaporated, and twice again redissolved in 1 mL of DzO followed by evaporation. This material was then dissolved in 0.5 mL of D20 and a drop of D20 containing 0.75% TSP was added prior to rewrding the the 'H NMR spectrum. Verification of stereospecificity may be aided by "spiking" the NMR sample with an addition of 1-2 mg of undeuterated NADH. Chemical shifts are determined relative to the internal TSP standard.
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Stereospecificity of Glucose Dehydrogenase 7 I I I I 2.9 2.8 2.7 2.8 2.5 Eight units of glucose dehy1 PPM drogenase were added to a reaction mixture containing 2.0 mL Figure 3. Spectraof NADH ~ ~ ~ & ~ H ] N (a)A Spectrum D H . O~&[~H]NADHfromglucosedehydrogenase of 7 mM NAD+and 40 mM Idcatalyzed reaction. (b) Spectrum of & ~ H ] N A D H from reaction with alcohol dehydrogenase catalyzed glucose in sodium phosphate reaction. (c)As for " b but including 1 mg of unlabeled NADH for internal comparison. (d) Spectrum of buffer (50 mM, pH 7.5) at 25 'C. NADH at pH 7.0. The progress of the reaction was monitored a s described above. Because the substrate from Sigma Chemical Co. Absorbance at 340 n m was meaand product of this reaction are non-volatile,it is necessary sured on a Beckman DU-64 spectrophotometer and N M R to separate the 4-[2H1NADH chromatographically. The mixture was loaded on a DEAE cellulose column (1x 15 spectra were collected on a Briiker AC 300 Fourier transcm) that had been wnverted to the bicarbonate form. Eluform NMR spectrometer. tion of the material proceeded using a 200-mL gradient of 0-0.5 M ammonium bicarbonate. Five-milliliter fractions Enzyme Assays were collected and monitored at 280 n m and 340 nm to Enzymes were assayed at 25 'C in 1-mL a w e s by meadistinguish between any residual NAD+,which is transsuring the increase in absorbance at 340 nm of solutions parent at 340 nm, and the desired 4-[2H]NADH, ~ ~ ~ mM NADt and 200 mM reduced substrate. with an absorbance of greater than .3 at 340 nm were Glucose dehydrogenase was assayed in sodium phosphate pooled and evaporated under reduced pressure. To help buffer (50 mM, pH 7.5) with glucose, and alcohol dehydroeliminate excess ammonium bicarbonate, the sample was genase was assayed at pH 8.5 in sodium phosphate buffer redissolved in 100 mL of 75% methanol in water. Approe(50 mM) with 2-propanol as the second substrate. The remately 2 g of dry ice was added, and the mixture was evapaction was initiated by the addition of NAD+. One unit of orated. Treatment with D20 proceeded as above, and the 'H NMR spectrum was recorded. activity is defined as the amount of enzyme that catalyzes I
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Results and Discussion Analysis of the results from this experiment depends uDon i n t e r ~ r e t a t i o nof t h e NMR sDectra of the 4['HINADH Groduced by the enzymatic reactions. The remaining unlabeled pro-R orpro-S hydrogen of deuterated NADH may be distinguished by two features. (1)Thepro-S hvdroeen amears u~fieldrelative to the ~ r o - Rhvdroeen " &d thLpro-s h$drogen shows great& chemical coud i n g to the oroton on C5. The NMR spectra resulting from the experi&ents described here, along with the spe&um of undeuterated NADH. are shown in Fimre 3. From Figure 3h it can be seen that alcohol dehydrigenase catalyzes the reduction of NAD' withpro-R specificity, transfering a hydrogen from the alcohol to the re face of NAD+.The peak is located above 2.7 ppm and is split into a doublet. This peak is due to thepro-S hydrogen, indicating that theproR position is deuterated. Note that the addition of undeuterated NADH, in spectrum 3c, c o d i s that the remaining peak is due to the more shielded of the two hydrogens. The sample of deuterated NADH prepared from glucose dehydrogenase, on the other hand, shows a single peak near 2.8 ppm corresponding to the pro-R hydrogen (Fig. 3a), indicating that the deuterium from ld-glucose has been transferred to thepro-S position. The lack of observable coupling to C5 also aids in the determination. The methodology described above is general and may be applied to a number of dehydrogenases for which deuterated substrates exist. This experiment also was tested l ! brockii, a pro-R with the alcohol dehydrogenase from ! specific NADP-dependent enzyme. NADPH required a gradient of 0 to 0.65 M ammonium bicarbonate for efficient purification, but may otherwise be handled similarly.
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This experiment introduces techniques in enzyme assays, chromatography, and high field 'H NMR. Wong and co-workers published a similar NMR experiment in this journal several years ago (3). Their experiment relied on a coupled exchange of the deuterium at the 4-position of the nicotinamide ring of NAD'. Analysis of stereospeciticity depends on the presence or absence of the peak corresponding to the proton a t C4 in the region of 9 ppm. This earlier ex~erimenteffectivelv introduces the concent of stercoselectlv~tyin dehydrogenases. However. the use of NMR in that work dws not reveal as much about molecular structure. In the experiment we have described, analysis of the high field 'H NMR sDenra touches on a varietv of interestingiopics in molecul& conformation, in addition to highlighting the stereospecificity of dehydrogenases. The central importance of NMR in chemistry, and the important function of dehydrogenases in biochemistry make this experiment worthwhile. Assaying an enzyme and performing the reduction with deuterated substrate can be accomplished easily in a single afternoon lab period. The DEAE column, which is necessary when non-volatile substrates are used, requires approximately 3 h to run and the DzO work-up an additional hour. Literature Cited 1. O@on,AG.Nohrr. 1848,162,963. 2. Kaapere*, G. J.: Pratt, R F J. Chem Educ. 1577,51 ,515416. 3. Lowrey, J. S.; Evans, T A . ;Won& S S. J Chem Edue. 1981.58, 816418 4. Fisher, H. F.;Ofofofof, P:Com. E.E.;Vennesland,B.; Westheimer,P H. J BioL Chem 1958,202,687-691.
5 . You,K . CRC C d t Re".Biochem. 18&1,17,313-451. 6. Bemer. S.A.Er.mripnt& lss8,385,623-637. 7. Oppenheimer, N J. J.A m Chem S a lW,105,3a325033. 8. Oppenheimer, N. J.,Amold, L.J. andKaplao, N. 0. Bioehem. 1978,17,2613-2619.
