Stereospecificity of NAD+/NADH Reactions A Project Experiment for Advanced Undergraduates Jonathan S. Lowrey, Ebaugh Laboratories. Denison University. Granville. OH 43203 Thomas A. Evans, Ebaugh Laboratories. Denison University. Granville. OH 43023 a n d S h a n S. Wong' University of Lowell. Lowell. MA 01854
Enzymes have achieved the status of "off-the-shelf' reagents. Their instructional applications now extend well beyond the biochemistry laboratory (1 2).The project described below is a study of enzymes dependent on the pyridine nucleotide coenzymes (Fig. la), the reactions of which are described in most organic chemistry and all biochemistry textbwks. It is a modification of work first reported by Kaplan, e t al. (3).T h e techniques required, e.g., working with microquantities, enzyme assay, UV-VIS speetrophotometry, and NMR spectrometry, are important in biochemistry but not unique to it. T h e project is appropriate for students in integrated laboratory programs, advanced organic chemistry, or biochemistry. Pyrldlne Nucleotldes a s Electron Carrlers Pyridine nucleotide coenzymes are important electron carriers in hiosynthesis and in energy-producing biochemical reactions (4). T h e reversible oxidation and reduction which they undergo (Fig. l b ) is equivalent to a reversible hydride transfer reaction. Taylor and Dixon (5)have described in this JOURNAL an affinity chromatography experiment based on lactate dehydrogenase (LDH), an enzyme which requires pyridine nucleotide coenzymes. Brown (6) has described experiments for the determination of coenzyme purity. A special nomenclature has been developed to accommodate the stereospecific transformation of NAD+/NADH. The enantiotopic hydrogens a t the 4-position of NADH have been labelled A and B a s shown in Figure lb. Enzymes which promote the transfer of HAare characterized as Class A enzymes; those for which H e is involved are identified as Class B enzymes. I t is also possible to describe the coenzyme transformation using more general stereochemical language. Class A enzymes add hydrogen to the re-face of NAD+ and promote transfer of the pro-R hydrogen of NADH (Fig. 2). Class B enzymes add hydrogen to the si-face of NADf and promote the transfer of thepro-S hydrogen of NADH. These reactions have been reviewed in a Van't Hoff-LeBel Commemorative Issue of Tetrahedron (7). Classillcation 01 Enzyme Stereospeclflcny The project involves the classification of enzymes according to their influence on the stereospecificity of the NAD+/NADH reaction. The general strategy is illustrated by the coupled reactions in Figure 3. Alcohol dehydrogenase (ADH), a Class A enzyme, catalyzes the formation of labelled NADH, which reacts with an oxidized substrate in a reaction mediated by an enzyme whose stereospecificity is "unknown." NMR analysis for the presence of H or D a t the 4-position of the pyridine ring allows conclusions to be drawn about the stePresented at the 12lh Central Regional Meeting of the American Chemical Society. May 1979. in Columbus. OH. Aulhw to whom inquiries should b+ sent. 818
Journal of Chemical Education
NAD
NADH (reduced form)
oxidized f o r m ) (b)
Figure I.(a) Srmmre of lboxWired form olnicotlnamide adenine dinucleotlde WAD+). and nicotinamida adenine dinucleotida phosphale (NADPt). (b)The NAD+/NADH reaction.
Flgve 2. (a) The nt- and si-facesof of NADH; Hs = pr0.S. HR = PI&
NAD+ (b) lb p r M and p r p S hydrogens
Nel
rsoction : CD3CDz0H
+
oxldozed
-
reduced
rubrtrote -~ubstrote
+
CD3CD0
Figure 3. Course of coupled enzyme reactions: M = H when EN22 is Class A. M = D when EN22 is Class 8.
IM
BD KLO
90
90
I1D
K)O
90
UO
Figwe 4. NMR of pyridine hydrogens of product NAD+.
reochemistrv of the second reaction. The progress of t h e n e t reaction is monitored b y following t h e disappearance of ouidized s u b s t r a t e hy a n indirect spectrometric method. Experimental procedures a n d results are given below for reactions coupling A D H with lactate dehydrogenase ( L D H ) a n d glutamate dehydrogenase (GDH). T h e n e t reactions are
00
1 11
ADH-LDH
CD,CD,OH + CH,CC-0-
in this buffer. Small sliquots (2.0ml) of NADH solution were placed in the spectrophotometer and a concentrated aqueous solution of oxidizing substrate appropriate to the enzyme assay was added to the cuvette via syringe. The final substrate roncentration in the assay mixture was aoorovimatelv 10 mM. For GDH activitv determinations the mixture was 20 mM in ammonium acetate. By adding only microliter volumes ofsubstrate solutions to the arsay euvette, the dilution effect on the NADH absorbance may be effectively ignored. The reaction was initiated by injection of sufficient enzyme solution to yield an initial rate of absorbance lossapproximating 0.5ARS units per minute. From the initial rate of decrease (AAImin), the enzyme activity may be calculated: AA.V.1 Activity (unitslmg protein) = 6.2. m where 6.2 mM-' em-' is the molar ahsorptivitv of NAnH at 340 nm. I is ihe cell path length. V is the milliliter volume ufthe NADH solution in the assay, and m is the amount of enzyme protein used in milligrams.
..
-
.
11
CD,CD,OH + Q,CCH,CH,C-C0,-
-
ADH-GDH
NH:
Demonstration of Enzyme Stereospecificity (Lab time 8 hours)
Experimental Procedures Materials and Equipment Enzymes and coenzymes for the experiment were obtained from Sigma Chemical Company. ADH was supplied as a solid. Both LDH and GDH were supplied as suspensions in buffered ammonium acetate solution. NADH and NADt were used in their sodium salt forms Both deuterium oxide (99.8%) and ethanol-d-6 (95% in D20) were purchased from Stohler Chemical Company. The microliter volumes necessary throughout the procedure were handled routinely using either a microsyringe or dispnsable calibrated capillary pipets (Aecupets). A Cary l18C recording UV-VIS spectrophotometer was used in coenzyme and substrate concentration determinations. All NMR spectra were recorded on a Varian T-60. Enzyme Assays (Lab time 3 hours) ADH, I,DH. and GDH were assayed by following the decrease of the NADH 340 nm nbsorhance in the following reartions (R.91: ADH + NADH -ethanol + NADt LDH pyruvate + NADH -+lactate + NAD+ CDH a-ketoglutarate + NHI+ + NADH -glutamate + NAD*
acetaldehyde
A standard 0.01 M phosphate buffer was Prepared for use with the enzyme systems by dissolving 1.36 g potassium dihydrogen phmphate in water, adjustingto pH 7.5 with sodium hydroxide,and diluting to one liter. A 0.15 mM solution of NADH salt and individual ADH, LDH, and GDH enzymesolutions of 1 mg proteinlml were prepared
I t was oossible to run two eauoled reaction exoeriments simultaneously. This parallel arrangement allowed comparison of the LDH and CDH reactions as well as an efficient use of laboratory time. (a) Loelole dehydrogenase. A 200-ml round-bottomed flask was charged with 150 mg (0.20 mmol) NADt, 45 mg (0.40 mmol) sodium pyruvate, and 30 ml of 0.01 M phosphate buffer, pH 7.5. After camplete dissolution of the salts, ADH (580 units) and LDH (300 units) were added to the mixture. The coupled reactions were then initiated by addition of 60 pl(0.80 mmol) ethsnol-d-6 and incubated a t room temperature to completion. The pH of the reaction mixture was checked periodically and adjusted to pH 7.54.0 with 25% NaOH. Careful regulation of the reaction pH is critical to the proper functioning of the enzymes. Denaturation will occur if the solution is allowed to become overly *";A;"
The progress of the net reaction was followed by assaying the pyruvate remaining in the system. Asmall sample (10pl) of the reaction mixture was added to a 2-ml aliauot of buffered 0.15 mM NADH in a soectroohotometer cuvette. he 340 nm absorbanceof the NADH was measured, and the assay reaction was rapidly driven tocompletion by addition of an excess amount of LDH enzyme (2-3 &its). A second absorbance reading was taken, and the amount of pyruvate remaining in the reaction flask was calculated from the total absorbancedecrease in the arsay procedure. This assay was performed prior to initiation of the LDH reaction and subsequently a t intervals during the first 2 to 3 hoursaf the reaction and again after 24 hours. The net reaction was considered complete when successive assays indicated no further decrease in the quantity of pyruvate; assay values of remaining pyruvate were plotted versus elapsed time. After reaction was complete the pH was adjusted to 2.5 with HNOs and lyaphylized by simple continuous evacuation with a standard mechanical pumpldry ice trap system. Approximately 5 ml deuterium oxide was added to the flask to redissolve the dry residue. Insoluble material was removed by centrifugation or filtration. The D20 soluVolume 58
Number 10
October 1981
817
tion was evaporated in uacuo to about I ml, and NMR analysis of the linal solution was performed. (b) Glutamole dehydrogenose. The procedure followed for the GDH reaction was completely analogous to that of the LDH experiment. A 200-ml r.b. flask was charged with 0.20 mmol NADt, 73.5 mg (0.40 mmd) n-ketoglutarate, 462.5 mg (6.0 mmol) ammonium acetate. and 30 ml phosphate buffer. After addition of ADH 1580 units) and GHD (50 units), the net reaction was initiated by addition of 5 0 4 ethanol-d-6 and inculrated at room temperature. Reaction progress was fallowed by the assay procedure described above, and GDH replaced LDH as the enzyme component. Following depletion of the n-ketoglutarate substrate the net reaction was quenched by acidification. Theresultinprnixture waslyophylized and treated with D20 as above. A l-ml deuterium oxide solution of the residue wasanalyzed by NMR. lc) NMR analysis. Acceptable 60 MHz spectra of unreaded NAD+ were obtained with 0.1 M NADf in deuterium oxide. Increased instrument gain isgenerally required todetect the pyridine 4-position proton (-96) critical to evaluation of the experimental results. Consequently, spectra are characterized by moderate noise. Spectra of solutions from the enzyme reactions exhibit a large aqueous proton signal approximately4.6 6 which can be reduced with completedrying in the lyophyliration step. Results and Dlscusslon Typical values from t h e enzyme assay were 96 unitslmg ADH, 530 unitslml L D H suspension, and 28 unitslml GDH suspension. In both the ADH-LDH and ADH-GDH reactions.
818
Journal of Chernlcal Education
the oxidized substrate was depleted within 24 hr. T h e NAD* produced in each reaction was compared t o a n authentic sample of NAD+ by analyzing t h e pyridine hydrogen N M R spectrum in the 8.10 6 region (10). T h e results a r e shown in Figure 4. T h e decrease in absorbance a t 9.0 6 in t h e NAD+ from the GDH reaction is evidence for deuterium incorporation and establishes GDH a s a Class R enzyme. T h e spectrum of NAD+ from t h e L D H reaction is identical t o t h a t of authentic NADC; L D H is a Class A enzyme. Both results a r e consistent with orevious reoorts in the literature (7). T h e work des&bed nhovk has heen performed hy students both individuallv and in oairs. It reauires flexihle a w s to the laboratory a n d a total time commitment of 10-15 hr. T h e centrality of NAD+/NADH in biochemistw and natureof t h e experiment amply justify such a commitment. Llterature Clted
141 Bruice. T . C.. and Ronkouie. S. J.. "Bioomsnic Mechaninrns." Val. 2. Chap. 9. W. A. Renjsmin. Now York. 1986. (51 Tay1ur.S. S.wd Dixon.J. E., J.CHEM. EoUc..55.675 119181. (61 Brown. P.H..J CHEM.RDIIC.. 53.98 119781. (7) Batterrhy,A. R..andStauntan, J.. lilrahedmn.30.17W 119741. J R i X ('hem.. 17S.RB5(19481. (81 H v r ~ t k ~ r . B . l ~ . . aKornherg.A.. nd 191 Sierel. J. M.. Mnnwomcry. C.A,. and H l a k . R. M.. Arch. Riochem. Biaphy.. 82.2% 119591. (10) Jadelzk.v. 0.. and Wede-Jamkdltrh..N. G..J. Rid. Chem..241. eS(1968).
