ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

The Alpha

166, 526-535 (19%)

Beta Epimerization

Nicotinamide NORMAN Department

Adenine

J. OPPENHEIMER2

of Chemistry,

University

AND

of California,

of Reduced’

Dinucleotide NATHAN

0. KAPLAN

San Diego, La Jolla, California

92037

Received July 1, 1974 The proton magnetic resonance spectra of the dihydronicotinamide ring of aNADH3 and the nicotinamide ring of aNAD+ are reported and the proton absorptions assigned. The absolute assignment of the C4 methylene protons of (YNADH is based on the generation of specifically deuterium-labeled (pro-S) B-deuterio-crNADH from enzymatically prepared B-deuterio-BNADH. The C4 proton absorption of aNAD+ is assigned by oxidation of B-deuterio-aNADH by the A specific, yeast alcohol dehydrogenase to yield 4-deuterioaNAD+. The epimerization of either (uNADH or BNADH yields an equilibrium ratio of approximately 9:l @NADH to aNADH. The rate of epimerization of (uNADH to BNADH at 38°C in 0.05, pH 7.5, phosphate buffer is 3.1 x 10m3 min-‘, corresponding to a half-life of 4 hr. Four related dehydrogenases, yeast and horse liver alcohol dehydrogenase and chicken M, and H, lactate dehydrogenase, are shown to oxidize aNADH to aNAD+ at rates three to four orders of magnitude slower than for BNADH. By using specifically labeled B-deuterio-LuNADH the enzymatic oxidation by yeast alcohol dehydrogenase has been shown to occur with the identical stereospecificity as the oxidation of BNADH. The nonenzymatic epimerization of aNADH to PNADH and the enzymatic oxidation (YNADH are discussed as a possible source of cyNAD+ in uioo.

(l-4). Subtle differences An enzymatically inactive oxidized nico- microorganisms tinamide adenine dinucleotide present in in the uv absorption spectra of CYNADH commerically available NAD+ has been and PNADH and the cyanide adducts of isolated and characterized by Kaplan et al. aNAD+ and PNAD+ (1, 3, 5) have been (1) as a pyridine coenzyme in which the reported and show that the absorptions of nicotinamide-ribose linkage has an (Y ano- the (Y anomers are characteristically shifted merit configuration as opposed to the p to longer wavelengths. The presence of configuration in the active coenzyme. This significant though variable amounts of dinucleotide, aNAD+, can consist of as (YNAD+ in extracts from a variety of orgamuch as 15% of the oxidized pyridine nisms has led to speculation both as to the coenzyme and is present in mammalian origin of cuNAD+ 4 and to its metabolic tissue extracts as well as in yeast and other function (l-5). The demonstration of a specific role for cyNAD+ or CXNADH, how‘This work was supported in part by grants from ever, has been elusive. Although there have the American Cancer Society (BC-60-P) and the been reports that aNADH can be oxidized National Institutes of Health (CA-11683). enzymatically (7-ll), this activity is nei1Present address: Department of Chemistry, Indither specific for CYNADH nor preferential ana University, Bloomington, IN 47401. with respect to PNADH. There has been no 3 aNAD+ and aNADH, the nicotinamide adenine case in which (YNADH has been shown to dinucleotides in which the nicotinamide-ribosidic be a specific coenzyme for any highly linkage has an (Y configuration; (4D)aNAD+, aNAD+ purified enzyme. with a deuterium label at the C4 position; IYNADD,, j3NADD,, (Y and BNADH with a deuterium the C4B position of the dihydronicotinamide Y.ADH, yeast alcohol dehydrogenase.

label in ring;

’ Recently the validity of some of these reports has been questioned and the artifactual nature of cuNAD+ proposed (6). 526

Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved.

a-8 EPIMERIZATION

An important clue to the source of the cy coenzymes has been provided by Woenckhaus and Zumpe (12) who have found that the aNADH thermally epimerizes to the enzymatically active PNADH. In conjunction with our investigations of the pyridine coenzyme conformat ion in solution, we have reported the reverse process. that /3NADH gradually acquires an (YNADH contaminant (13). Here we report studies under approximately physiological conditions of the equilibration between cy and p forms of YADH, data on the enzymatic activity ofcvNADH with four common dehydrogenases and the stereospecificity of the enzymatic oxidation of LYNADH by yeast ADH. METHODS

Preparation

of

Materials

The dinucleocides fiNAD+, PNADH, and nNAD+ were obtained from P-L Biochemicals and were used without further purification. aNADH was prepared by dithionite reduction of nNAD+ (14) and was purified on a DE:AE~cellulose column eluted with an ammonium bicarbonate gradient (0.005 M to 0.5 M); the preparation had less than 3%~NADH contamination as determined by enzymatic assay. Yeast and horse liver alcohol dehydrogenase (EC 1.1.1.1) were purchased from :P-L Biochemicals and the crystalline chicken M, and chicken H, lactate dehydrogenases (EC 1.1.1.27) were prepared by Mr. F. Stolzenbach (15).

Enzymatic

ActivitJf

The activity of aNADH with alcohol dehydrogenase was measured in 0.1 M phosphate buffer at pH 7.5, with 0.1 M acetaldehyde and either 0.1 mM@NADH or aNADH. The activity of aNADH with lactate dehydrogenase was measured under the same conditions except that the 0.1 M acetaldehyde was replaced with 10 mM pyruvate. The oxidation of aNADH could be initiated either by addition of oxidized substrate to cuNADH/dehydrogenase or dehydrogenase to aNADH/oxidized substrate, No reaction was observed with aNADH and dehydrogenase in the absence of substrate or with the wrong substrate. e.g., pyruvate. aNADH, and YADH gave no observable decrease in the 340.nm absorption.

Epimerization The rate of epimerization BNADH in 0.05 M, pH 7.5, buffer was measured at 38°C. BNADH in the ~uNADH sample 340-nm absorption which could

of 3 mM aNADH or potassium phosphate The concentration of was determined as the be rapidly discharged

527

OF NADH

by assay levels of yeast alcohol dehydrogenase/acetaldehyde. The residual absorption of 340 nm after addition of yeast alcohol dehydrogenase/acetaldehyde to the PNADH sample was measured and assumed to reflect the nNADH content. The concentration of the enzyme used for these determinations was insufficient to cause any significant oxidation of aNADH.

Synthesis

of 4B-deuterio

aNADH

Specifically labeled (uNADH with deuterium in the C4B position, aNADD “, was synthesized from 300 mg of PNADD H prepared by lipoyl dehydrogenase reduction in D,O (0). The fiNADD H was dissolved in 20 ml of 0.1 M ammonium bicarbonate buffer, pH 8.5, and incubated in boiling water for 15 min. The solution was then cooled in an ice bath. The BNADD,, was oxidized by yeast alcohol dehydrogenase/acetaldehyde, with care taken to insure that the amount of enzyme used would cause negligible oxidation of oNADD,,. The solution was placed on a DEAEcellulose column and eluted with a linear ammonium bicarbonate gradient. The aNADDs was clearly resolved from the deuterium-labeled (4D)BNAD+. Approximately 8? aNADD s was recovered as well as a nearly equal amount of the specifically labeled primary acid product (16). The otNADD s structure was confirmed by the lack of observable enzymatic activity with assay concentrations ofyeast alcohol dehydrogenase, and a red shifted UV absorption maximum at about 342 nm and from the PMR spectrum.

SpecificitS,

of Enzymatic

Oxidation

(4D)crNAD’ was prepared by the enzymatic oxidation of oNADD,. The oNADD, was shown to be free of oxidized pyridine coenzyme prior to enzymatic oxidation by the failure to detect the formation of any cyanide adduct after addition of KCN. Twenty milligrams of yeast alcohol dehydrogenase was added to 8 ml of a 5 mM aNADDs in 0.1 M ammonium bicarbonate, pH 8.5. with 0.1 M acetaldehyde. The absorption at 340 nm was followed until no further decrease was observed. The solution was then placed on a DEAE-cellulose column and eluted with a linear ammonium bicarbonate gradient. The (4D)aNAD+ was recovered and gave a 331.nm absorption upon addition of cyanide. The configuration of the nucleotide and the presence of the deuterium label were confirmed by comparison of the pmr (proton magnetic resonance) spectrum with the spectra of oNAD+ and fiNAD*.

Spectral Determinations Proton magnetic resonance spectra were recorded on a Varian Associates field sweep HR-220 proton magnetic resonance spectrometer and the signal-tonoise ratio was enhanced with a Nicolet 1074 computer. Homonuclear spin decoupling was performed using a Wavetek 131A voltage-controlled oscillator.

528

OPPENHEIMER

AND KAPLAN

TABLE

I

CHEMICAL

Reduced coenzymes BNADH aNADH

70 mM 70 mM

Oxidized coenzymes fiNAD+ 90 mM oNAD+ 70 mM

SHIFTS”

PC6

PC1’

PC5

PC4HA

PC4HB

1344.5 1345.5

1317.0 1285.0

1056.0 1094.0

1050.0 1022.5

602.5 605”

582.5 595b

PC4

AC8

PC5

AC2

PCl’

ACl’

1951 .o 1935.0

1853.0 1851.5

1813.5 1776.0

1780.0 1768.0

1349.5 1409.5

1324.0 1315.5

AC8

AC2

PC2

1862.0 1865.5

1797.0 1798.5

1525.5 1521.0

PC2

PC6

2660.0 1995.5

2023.0 1959.0

ACl’

a Chemical shifts in Hz from TSP at 220 MHz, 22°C. The chemical shift values are reported to within 0.5 Hz. “The chemical shift nonequivalence of the PC4 protons of NADH was determined from the nonspecific deuterium-labeled aNADH (13).

about 10 Hz nonequivalent and appear as a very strongly coupled AB pattern (11). The chemical shifts for the C6 and C2 protons also differ for the two anomers and can be easily distinguished in a mixture of CYNADH and PNADH. The results of incubation of a solution of aNADH and PNADH at 90°C are shown in RESULTS Fig. 2. The single absorption peak for the (uC4 methylene protons is dramatically Spectral Analysis transformed after incubation at 90°C for 16 The chemical shifts of the proton absorpmin into a four-peak asymmetric AB pattions of the dihydronicotinamide ring, tern. For the pure PNADH sample the nicotinamide ring, and the ribose C-l’ effects are more subtle. After a 20-min protons of PNADH, PNAD+ and aNAD+ incubation at 9O”C, the nearly symmetriare listed in Table I. The pmr spectra of cal AB quartet of the PC4 methylene pro(YNADH and PNADH are shown in Fig. 1. tons has become markedly asymmetric The PC2, PC6, PC5, and PC4 protons were with the downfield central peak increasing assigned by the similarity in chemical in height and area.6 As can be seen, the shifts and coupling constants with the pmr spectra of the initially pure samples of corresponding protons in PNADH (17, 131 PNADH and aNADH have become virtuand were confirmed by homonuclear spin ally identical after incubation at 90°C. decoupling, although the absolute assignWe have also observed that solutions of ment of the &4 protons cannot be deter- PNADH, which have been stored at ~ 20°C mined solely on this basis. The spectra in in sealed tubes under argon, develop addiFig. 1 show distinct differences in the tional proton absorptions corresponding to absorption patterns for the C4 methylene the formation of an aNADH contaminant and ribose proton regions.5 The chemical after a few months. The appearance of an shift nonequivalence of the PC4 protons is asymmetric AB pattern for the PC4 methabout 20 Hz at 220 MHz and they appear ylene protons and an increased area under as an A3 quartet (18, 191. By comparison, the downfield central peak of this pattern the C4 protons of the u anomer are only Samples were twice lyophilized from 99.8% D,O and run in 100% D,O, pD of 8.5 and 22”C, the ambient temperature of the probe. Sample volume was 0.25 ml and Teflon vortex plugs (Wilmad) were used. An internal standard, 3 mM TSP trimethylsilyl sodium propionate (tetradeuteriol, was used and 1 mM EDTA was added to suppress line broadening from possible paramagnetic impurities.

5A complete assignment of the ribose protons and conformational analysis of both aNAD+ and aNADH as they compare to the B coenzymes are beyond the scope of this publication and will be discussed in a forthcoming paper.

6The AB pattern of the PC4 methylene protons is intrinsically asymmetric due to the unequal coupling of the C5 proton to the A and B protons. The presence of (rNADH contaminant serves to accentuate this by absorbing at the same frequency as the downfield central peak of the AB pattern of BNADH (13).

529

Hz from TSP 01 220 MHZ

FIG. 1. Pmr spectra of BNADH and (YNADH in D,O, pD 8.5, 22°C. Chemical shifts are in Hz from TSP at 220 MHz. The adenine protons are designated by the letter A, the pyridine protons by

FIG. 2. The C2, C6, and C4 dihydronicotinamide ring proton absorptions of the pmr spectra at 22”C, in D,O, pD 8.5. Chemical shifts are in Hz from TSP at 220 MHz. (a) lpure BNADH; (b) BNADH after incubation at 90°C for 20 min; (cl aNADH after incubation at 90°C for 16 min; (d) (YNADH after incubation at 90°C for 10 min; (2) pure uNADH.

has been previously observed by Sarma and Kaplan (1.8) in pmr studies of PNADH samples which had not been heated. However, the presence of an cvNADH contami-

nant was not suspected in this earlier study. Absorptions due to the C2 and C6 protons also appear concomitantly with the changes in the C4 methylene group. In the spectrum of PNADH it is difficult to detect the absorptions of the oC2 and ctC6 protons which are broad and tend to become lost in the base line noise. However, by computer averaging, the presence of these peaks can be unmistakably demonstrated. As the pmr studies show, samples of (uNADH and (3NADH become nearly identical after a brief incubation at 9O”C, and reveal about a 9:l predominance of the /3 anomer. Consistent with this result is the observation that about 90% of the uv absorption at 340 nm of these heat-treated samples can be rapidly discharged by yeast alcohol dehydrogenase/acetaldehyde at pH 7.5 and that the residual absorption corresponds to about 10% of the original absorption and has a X,,, at 343-344 nm, the absorption maximum for aNADH. Thus, the pmr results establish that the 340-nm absorption remaining after enzymatic oxidation of PNADH which has been heated or stored for long periods of time reflects LuNADH. Assignment

of the

C4 Methylene

Protons

Specifically labeled (UNADD, prepared by the thermal epimerization of PNADD, has a one-proton absorption at 607 Hz corresponding to the C4HA proton. This is

530

OPPENHEIMER

the value of the chemical shift for the downfield proton in the unresolved (uC4 methylene A-B pattern. Thus, the pmr absorptions of the methylene protons in (uNADH are assigned to the absolute configuration of the dihydronicotinamide ring; the downfield proton at about 605 Hz is the A proton (pro-R configuration) and the upfield proton at 595 Hz is the B proton (pro-S configuration). The relative order of the chemical shift of the C4 protons is maintained for both aNADH and BNADH; the A proton is downfield and the B proton is upfield. Since the origin of the observed chemical shift nonequivalence of the C4 protons in PNADH and reduced NAD+ analogs (21) is apparently due to a fast, preferential intramolecular association of the B face of the dihydronicotinamide ring with the adenine ring (13), it would seem reasonable to suggest that the similarity in the chemical shift pattern of crNADH is likewise due to a B side interaction with adenine. This does not mean that the over-all tertiary structure of aNADH is identical to that of PNADH. On the contrary, CD studies have shown a marked difference in the relative orientations of the dihydronicotinamide chromophores with the adenine chromophores in PNADH and aNADH (22). However, our results do indicate that although the relative orientation of the rings may differ, as would be expected from the differences in geometry, the same side of the dihydronicotinamide moiety is apparently interacting with the adenine moiety.

AND KAPLAN

For incubations longer than 12 hr, the concentration of primary acid product becomes greater than 20% of the original dihydronicotinamide. For a reaction which does not proceed to completion, the observed rate constant for the forward reaction is not equal to the true rate constant. However, the correction here would be small and thus would affect the important result that the half-life of 4 hr for the reaction in vitro at 38°C would be physiologically quite significant. The effect of incubation of PNADH at 38°C is less distinct. There is a marked increase with time of the absorption at 340 nm remaining after oxidation of the /3NADH with alcohol dehydrogenase (Fig. 3). Based on the pmr results for incubation of PNADH at 9O”C, this residual uv absorption at 340 nm is assumed to reflect aNADH. Accurate kinetic data are not available directly for the PNADH epimerization because of the inherent error in measuring the small concentration of crNADH in the presence of large concentrations of PNADH. However, this rate constant can be estimated as 3.2 x 1O-4 min-’ using an approximate equilibrium constant of 10 and the cvNADH to PNADH rate constant. Enzymatic

Activity

The relative rates of enzymatic oxidation of cvNADH were determined for the follow-

Epimerization The time course of the conversion of (uNADH to PNADH is shown in Fig. 3. This reaction shows typical first-order kinetics with an observed rate constant of 3.1 x -o-----o ------1O-3 min-’ at 38°C. The calculated first0 2 4 TIM," 8 IO 12 ill,) order rate constant has not been corrected FIG. 3. The epimerization of 3 mM aNADH or for the contribution from the reverse reaction. The equilibrium constant at 38°C for @NADH at 38°C in 50 AM, pH 7.5 potassium phosphate. The concentration of ~YNADH f-1 was the epimerization can only be estimated measuredas the percent of the A,,, which could not because of the formation of primary acid be discharged by yeast alcohol dehydrogenase/acetalproduct (16). While the primary acid prod- dehyde. The concentration of uNADH in the BNADH uct has no uv absorption at 340 nm, its sample (-----) was measured as the percentage of formation prevents the establishment of an residual absorption at 340 nm after enzymatic oxidaequilibrium for the epimerization reaction. tion of the BNADH.

o-j3 EPIMERIZATION

ing enzymes; chicken H, and chicken M, lactate dehydrogenase and horse liver and yeast alcohol dehydrogenase. The ratios of the decrease in absorption at 344 nm of 0.1 IIIM aNADH to the rate of oxidation of 0.1 IIIM PNADH with identical substrate and buffer concentrations are listed in Table II. A small but significant enzymatic activity is observed with the cy anomeric form of NADH. The reported relative rates are a qualitative comparison of the activity of (uNADH with respect to PNADH as cofactors in the enzymatic reduction of the oxidized substrate. The K, of the aNADH is illustrated in Fig. 4 and indicates a rather strong binding to yeast ADH. The product of (uNADH reaction was determined to be aNAD+ by the addition of cyanide which produced a 332-nm uv absorption maximum as opposed to a maximum at 327 nm for the cyanide adduct of PNAD (1, 3, 4). Also, the oxidized nucleotide formed was enzymatically inactive at TABLE RELATIVE

II

RATES OF OXIDATION

OF (YNADH

AND

PNADH

V"N ma 13.1 x lo-’

Chicken H, lactate dehydrogenase Chicken M, lactate dehydrogenase Horse liver alcohol dehydrogenase Yeast alcohol dehydrogenase

0.87 x lo-’ 3.5 x lo-’ 8.0 x lo-’

o V,,,,,, is the ratio of the rates of oxidation of aNADH to that of BNADH using the standard assay conditions outlined in the methods section. I

I

I

IO- YEAST ADH

0 Kmapp=OJ4mM

IO

20

30

I LZNADHI

40

50

nM.1

FIG. 4. Double-reciprocal plot of the dependence of the initial velocity of acetaldehyde reduction on aNADH concentration for yeast alcohol dehydrogenase. The apparent K, of (YNADH is 0.34 mM. Measurements were made in 0.1 M phosphate buffer, pH 7.5, and 0.1 M acetaldehyde.

OF NADH

531

pH 10.5 with the reduced substrate under conditions which readily reduced PNAD+. Finally, the pmr spectrum of the isolated oxidized pyridine coenzyme after extraction confirmed the product was (rNAD+. Our observations as well as those of others involved in the work on the inactivity of aNAD+ towards enzymatic reduction leads to the contention that the oxidation of aNADH under typical assay conditions can be considered irreversible. Because of the low redox potentials of PNADH and (wNADH, aNADH is even 20 mV more negative than PNADH (2); their oxidation is strongly favored at pH 7.5. Furthermore, the enzymatic rate of oxidation of (uNADH is three to four orders of magnitude slower than for PNADH. Thus, the rate of reduction of cuNAD+ under the stated conditions would be still slower because of the unfavorable equilibrium and redox potential, making the oxidation of aNADH essentially irreversible. That is, while the reduction of aNAD+ is expected from consideration of microscopic reversibility, the rate at pH 7.5 would be unobservable because of the unfavorable equilibrium and the slow rate. Stereospecificity of the Enzymatic tion of CYNADH

Oxida-

Specifically labeled (uNADH with deuterium in the B position, aNADDs, was oxidized by yeast alcohol dehydrogenase and the pmr spectra of the recovered aNAD+ is shown in Fig. 5. Yeast alcohol dehydrogenase is an A-specific enzyme; it transfers hydride to or from the A side of the nicotinamide ring of the coenzyme. Thus, if aNADD,, were oxidized with the same stereospecificity as PNADH, then the transfer of the A proton to the substrate should yield an a-nicotinamide ring with a deuterium incorporated at the C4 position. As can be seen from Fig. 5, the doublet at 1935 Hz is missing; hence, the oxidation of the 4B-deuterio dihydronicotinamide moiety of aNADD, occurs with the identical specificity as for PNADH. The absence of that proton absorption in (4DlaNAD+ also provides an unequivocal assignment of the C4 nicotinamide proton of cuNAD+ and by conventional coupling-constant analy-

532

OPPENHEIMER is00 +

1400 ,

13po

I

ccNAD+ PCLH

,

,,ACEH

ACBH,

PCfH

ACI'H

(4DlaNAD+ I

I’ 2000

I 1900 Hz from

1

,I’ 1800 TSP

01220

m MHL

FIG. 5. Pmr spectra of PNAD+ and cuNAD+ in D,O, pD 8.5, and 22°C. (a) 90 mMflNAD+; (b) 70 mM aNAD+; (c) 25 mM (4D)aNAD+ prepared by the enzymatic oxidation of crNADD, by the A specific enzyme Y.ADH. The lack of the doublet at 1935 Hz and the conversion of the PC5 proton absorption at 1776 Hz from a triplet to a doublet is conclusive evidence for the stereospecific removal of the PC4 proton from aNADD, by Y.ADH. The differences in the chemical shift of the AC8 and AC2 protons in spectra (b) and (c) reflect effects of the concentrationdependent association of the adenine moieties of aNAD+, which is also observed in aNAD+ (29). sis, the remaining proton absorptions unambiguously assigned.

are

DISCUSSION

Enzymatic Activity The observation that cvNADH has some enzymatic activity raises questions about how a dehydrogenase “designed” for ,8NADH cannot only bind the Q anomer but utilize it as well. As shown in Fig. 4, yeast alcohol dehydrogenase has an apparent K, of 0.34 mM for CINADH, a value which suggests comparatively strong binding. This apparent K, is about seven times larger than the K, for PNADH (24) but considerably smaller than the K, for either AMP or adenine ribose pyrophosphate ri-

AND KAPLAN

bose (25). Thus, the greatly diminished relative rate of oxidation of aNADH compared to PNADH can probably be attributed to a decrease in V as a result of different geometry and orientation of the dihydronicotinamide ring in the active site of the dehydrogenase. The fact that CYNADH has any activitv at all, let alone the identical stereospecificity of oxidation, means that sufficient degrees of conformational freedom exist for the bound (Y coenzyme to permit the specific orientation of the dihydronicotinamide ring for reduction of the substrate. This result is consistent with the ability of 5’-AMP to bind to dehydrogenases while PNMN+ and PNMNH do not-that is, it suggests that the dehydrogenase binds the adenineribose-phosphate portion specifically while only specifying the seating of the pyridine ring and not the pyridine ribose. Thus, aNAD+ would bind with the adenine moiety seated properly but with the pyridine ribose out of alignment but yet still maintaining the ability to allow access of the pyridine ring to the active site for the hydride transfer.

Source

of

NAD+

In the study of the epimerization of a-pyridine coenzymes, Woenckhaus and Zumpe could find no evidence for the direct thermal epimerization of aNAD+ and we have confirmed their observation. However, PNAD can be recovered from the cyanide adduct of aNAD+ which has been heated (12). Their result demonstrates that the NAD-cyanide adduct is also capable of epimerizing and suggests that dihydronicotinamide-ribose moieties are susceptible to epimerization. The observed epimerization is assumed to occur via the same mechanism as the acid-catalyzed hydrolysis of nucleosides (26. 27); the ribose ring opens, a double bond is formed, and a positive charge is generated on the N-l nitrogen, but instead of attack by water, the ribose ring recloses, as shown in Eq. PI.

a-8 EPIMERIZATION

The oxidizeId coenzymes, cuNAD+ and ,8NAD+, cannot epimerize by this mechanism because the ring nitrogen which already carries a positive charge is unable to donate electrons to form the double-bond intermediate. The electron-rich. uncharged dihydronicotinamide ring of NADH or the NAD-cyanide adduct can donate electrons to form this double-bond intermediate. and therefore, they would be expected to e.pimerize as has been observed (12, 13). The inability of the oxidized pyridine coenzymes to epimerize raises questions as to the origin of oNAD+ in U~UO.If aNAD+ cannot directly epimerize, then the only apparent source of the cy coenzyme is the oxidation of tvNADH formed by the epimerization of PNADH. The results shown in Fig. 3 indicate that the epimerization of PNADH is sufficiently rapid to generate physiologically significant concentrations of (YNADH wjthin hours. For warm-blooded animals (- 37”C), PNADH would reach the 9:l equilibrium mixture with aNADH in a period of less than 1 day. Thus, the nonenzymatic epimerization of PNADH shown in vitro could provide a constant level of aNADH in uiuo barring any enzymatic regulation of the epimerization. The previous reports (5-9) of the enzymatic oxidation of aNADH, in addition to our data on its activity with four common dehydrogenases, suggest that adequate capacity exists in uiuo for the oxidation of (rNADH to QNAD+~. In view of the unfavorable equilibrium for the enzymatic reduction of aNAD+, the oxidation of cyNADH can be treated as being irreversible. Thus, the formation of aNAD+ in uiuo would be a “dead end” reaction, resulting in the net accumulation of significant concentrations of aNAD+. The reports that concentrations of aNAD+ in organisms and tissues may be greater than 10% of the PNAD+ levels (1, 3) indicate that the 7 The physiological concentrations of dehydrogenases are so high ( >2 r&gram tissue) that it appears reasonable that any cvNADH generated would be quickly oxidized by dehydrogenases to cuNAD+ even though the specific activity for oxidation of the aNADH is very low.

533

OF NADH

concentration of oNAD+ in some cases may equal or exceed that of NADP+. The steady-state concentration of aNAD+ that could be generated by our postulated mechanism would be a function of: (1) the rate of formation of aNADH which will depend on pH, temperature, ionic strength, and the concentration of PNADH; (2) the rate of enzymatic oxidation of (uNADH to aNAD+; and (3) the rate of catabolism or excretion of aNAD+. The levels of aNAD+ could also be controlled if there were specific aNAD+ nicotinamideglycohydrolases, aNAD+ epimerases, or aNAD+-specific reductases. There is as yet no evidence for the existence of any of these enzymes. The following scheme would then be suggested for the origin of aNAD+ in living cells. BNADH

nonenzymatic

+ aNADH

dehydrogenases,

oxidases

+ aNAD+

The aNAD+ would then be isolated by acid extraction. This scheme would predict that the concentration of cuNAD+ would tend to follow, after a period of time, any changes in the over-all concentrations of the pyridine coenzymes. Such behavior has been ovserved in the rat by Ricci et al. (4). Injections of nicotinic acid into rats results in a rapid increase in the concentration of PNAD+ in erythrocytes and liver, followed by a subsequent increase in the concentration of aNAD+. From these studies it appears that the /3 form is synthesized and then a slow conversion to the (Y form occurs. It has been recently suggested that the presence of aNAD+ might be an artifact generated solely by the procedure for the isolation of the pyridine coenzymes; extraction with hot ethanol causing the thermal epimerization of PNADH to aNADH and a subsequent nonenzymatic hydride transfer from aNADH to ,BNAD+, yielding the observed aNAD+ (6). The two steps in this mechanism differ in one critical aspect. The epimerization of PNADH to cvNADH can be considered an unimolecular reaction, if the pH dependence is neglected, while the hydride trans-

534

OPPENHEIMER

fer reaction from aNADH to oNAD+ is a bimolecular reaction. Thus, the rate of epimerization of PNADH is independent of the initial concentration while the rate of hydride transfer will depend on the concentration of the reactants as has been observed by Ludoweig and Levy (28). Extrapolating from the rates we report for the epimerization, it is quite probable that BNADH will equilibrate with aNADH under the conditions for extraction outlined by Suzuki et al. (3). However, it is not clear to what extent the hydride transfer will proceed because of the low concentrations of the pyridine nucleotides in the extract. Thus, the total yield of aNAD+ will depend on the length of time the extract is allowed to incubate as well as on the concentrations of the reacting nucleotides. Admittedly, this mechanism could be the source of significant concentrations of cvNAD+ and may account for the presence of aNAD+ in hot ethanol extracts of organisms and tissues, but it cannot account for the reports of aNAD+ from organisms and tissues which have only been subjected to extraction with trichloroacetic acid or perchloric acid (1, 4). Thus, there remains solid experimental evidence for the natural occurrence of aNAD+. In addition, the rate of hydride transfer from (YNADH at 37°C giveLl the physiological concentrations of PNAD (28) would be many orders of magnitude slower than the observed rates compared to the enzymatic oxidation of (rNADH; this might suggest that the mechanism of Jacobson et al. (6) may not contribute significantly in the in uiuo synthesis of aNAD+. Our results suggest that a plausible mechanism for the continuous synthesis of aNAD+ could exist in uiuo based on the generation of aNADH by the nonenzymatic epimerization of BNADH, and the subsequent enzymatic oxidation of the aNADH to aNAD+. One might expect a greater build-up of aNAD+ than has been reported in animal or microbial extracts if the oxidized CYnucleotide is a dead-end product; however, the possibility exists that most of the /3NADH in cells is protein bound (2), although there is recent evidence against this hypothesis (29), and thus,

AND KAPLAN

might not be susceptible to epimerization. It remains to be seen whether the aNAD+ or L~NADH which can be produced from PNADH by the sequence of reactions discussed here has any significant biochemical function. At present there are no indications of any physiological role for the (Ypyridine nucleotides. Nevertheless, the presence of aNAD+ is intriguing in that its origin may represent a nonenzymatic process either in uiuo or in uitro. ACKNOWLEDGMENTS The authors thank Drs. L. J. Arnold, Jr. and E. H. Cordes for helpful discussions and advice. REFERENCES 1. KAPLAN, N. O., CIOTTI, M. M., STOLZENBACH, F. E., AND BACHER, N. R. (1955) J. Amer. Chem. Sot. 77, 815. 2. KAPLAN, N. 0. (1960) in The Enzymes (P. D. Boyer, H. Lardy, and K. Myrback, eds.), Vol. III, Academic Press, New York. 3. SUZUKI, S., SUZUKI, K., IMAI, T., SUZUKI, N., AND OKUDA, S. (1965) J. Biol. Chem. 240, PC 544. 4. Rrccr, C., PALLIN], V., AND MARTELLI, P. (1965)

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Biophys. Res. Commun. 19, 296. G., AND WOENCKHAUS, C. (1965) Ann. Chem. 690, 170. JACOBSON, E. L., JACOBSON, M. K., AND BERNOFSKY, C. (1973) J. Biol. Chem. 248, 7891. CI.ARK, W. M., KAPLAN, N. O., AND KAMEN, M. D. (1955) Bocteriol. Reu. 19, 234. OKAMOTO, H. (1973) Biochem. Biophys. Res. Commun. 50, 793. OKAMOTO, H. (1971) Methods Enzymol. 18B, 67. OKAMOTO, H., ICHIYAMA, A., AND HAYASHI, 0. (1967) Arch. Biochem. Biophys. 118, 110. SUZUKJ K., NAKANO, H., AND SUZUKI, S. (1967) J. Biol. Chem. 242, 3319. WOENCKHAUS, C., AND ZUMPE, P. (1965) Biochem. Z. 343, 326. OPPENHEIMER, N. J., ARNOLD, L. J., JR. AND KAPLAN, N. 0. (1971) Proc. Nat. Acad. Sci. USA 68, 3200. LEHNINGER, A. L. (1957) Methods Enzymol. 3, 885. PESCE, A., FONDY, T. P., STOLZENBACH, F. E., CASTILLO, F., AND KAPLAN, N. 0. (1967) J. Biol. Chem. 242, 2151. OPPENHEIMER, N. 3. (1973) Biochem. Biophys. Res. Commun. 50, 683. MEYER, W. L., MAHLER, H. R., ANDBARKER, R. H., JR. (1962) Biochim. Biophys. Acta 64, 353. PATEL, D. J. (1969) Nature (London) 221, 1239. SARMA, R. H., AND KAPLAN, N. 0. (1969) J. Biol. Chem. 244, 771.

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OF NADH

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26. CAPON, B. (1969) Chem. Reu. 69, 407. 27. DEKKAR, C. A., AND GOODMAN, L. (1970) in The Carbohydrates-Chemistry and Biochemistry (Pigman, W., and Horton, D., eds.), Vol. IIA, p. 43, Academic Press, New York. 28. LUDOWIEC, J., ANDLEVY, A. (1964) Biochemistry 3, 373. 29. BERNOFSKY, C., AND PANKOW, M. (1973) Arch. Biochem. Biophys. 156, 143. 30. MCDONALD, G., BROWN, B., HOLLIS, D., AND WALTER, C. (1972) Biochemistry 11, 1920.