A CHEMICAL REACTION OF HYDROXYLAMINE WITH DIPHOSPHOPYRIDINE NUCLEOTIDE

A CHEMICAL REACTION OF HYDROXYLAMINE WITH DIPHOSPHOPYRIDINE NUCLEOTIDE*

BY ROBERT MAIN BURTONt AND NATHAN 0. KAPLAN

(From the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland)

(Received for publication, March 17, 1954)

Kaplan and Ciotti (1) reported that hydroxylamine would inhibit liver and yeast alcohol dehydrogenases. Studies of this inhibition were extended by Kaplan et al. (2, 3), and reaction mechanisms were postulated for the hydroxylamine inhibitions; the essential feature of the hydroxylamine inhibition is the enzymatic formation of a DPN’-hydroxylamine compound that is bound to the enzyme (2, 3).

DPN has been shown to react chemically with a number of reagents: alkali (4), cyanide and bisulfite ions (5, 6), dihydroxyacetone (7, S), and sodium dithionite (9). These reactions are all attacks of a nucleophilic reagent upon a positively charged center. Since hydroxylamine reacts as a nucleophilic reagent in both acidic and basic solutions in oxime formation (lo-14), it was suggested that a chemical reaction between hydroxylamine and DPN may occur. Previous attempts to show a chemical reaction were unsuccessful (3, 6). However, in view of recent studies on DPN addition reactions (8), it was thought that the conditions used previously to show a DPN-hydroxylamine reaction were too mild. This paper contains a description of the conditions necessary for the chemical reaction between hydroxylamine and DPN. A reaction mechanism is postulated, and a discussion of the chemical reaction is presented in rela- tion to the mechanism of action of alcohol dehydrogenase. Equation 1 formulates the over-all chemical reaction of DPN with hydroxylamine.

DPN+ + NH20H + DPN-NHOH + Hi- m

* Contribution No. 81 of the McCollum-Pratt Institute. Aided by grants from the Rockefeller Foundation, the American Cancer Society as recommended by the Com- mittee on Growth of the National Research Council, and the Williams-Waterman Fund.

t Research Fellow of the National Heart Institute, United States Public Health Service.

1 The following abbreviations are used: diphosphopyridine nucleotide, oxidized form, DPN, reduced form, DPNH; addition compound of DPN and hydroxylamine, DPN-NHOH. AZ3 represents the optical density of the solution, as described, in a cell with a light path of 1 cm.; i.e., AE = - loglo (I solution)/(Z solvent) where Z is the intensity of the light emerging from the cuvette.

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448 HYDROXYLAMINE REACTION WITH DPN

Material and Methods

Hydroxylamine hydrochloride was purchased from the J. T. Baker Chem- ical Company, methoxyamine from the Eastman Kodak Company, and the monoethylamine and diethylamine were gifts of Sharples Chemicals, Inc. DPN was a z “90” preparation purchased from the Sigma Chemical Com- pany, and the solutions of DPN were assayed either by the cyanide method (6) or with yeast alcohol dehydrogenase and ethanol. Crystalline yeast alcohol dehydrogenase was prepared by the method of Racker (15). All

0.60 - ; 0.40 -

W a

0.20 - 5

HYDROXYLAMINE CONCENTRATION

(M/L) FIG. 1. The effect of hydroxylamine concentration. 0.033 M sodium pyrophos-

phate and varying amounts of hydroxylamine as indicated were adjusted to pH 10.5 with either sodium or potassium hydroxide. The final volume was 2.9 ml. A small volume of DPN solution was added to make a final concentration of 1.67 X 1OW M and the increase in optical density at 315 rnp recorded.

spectrophotometric measurements were made with the model DU Beck- man spectrophotometer. All pH measurements were made with the model G Beckman pH meter. The solutions containing the amines were always adjusted to the indicated pH immediately prior to running the reactions; hence the change in concentration of the amine by the decomposition of the hydroxylamines or by volatilization of the alkyl amines at the high pH used will be minimized for the time interval of the reaction.

Results

Effect of Hydroxylamine Concentration-The chemical reaction of DPN with hydroxyiamine requires a high hydroxylamine concentration. Fig. 1


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R. M. BURTON AND N. 0. KAFLAN 449

shows the effect upon the equilibrium position2 of the reaction formulated in Equation 1 and of increasing the concentration of hydroxylamine from 3 X 1Cr2 to 1.3 M. The rate of the reaction even at low concentrations of reactants is extremely rapid, and hence the equilibrium point is reached before a spectrophotometric reading can be made (5 to 15 seconds). That this is an equilibrium position is demonstrated by the effect of changing

W

2

3.0

2.0

1.0

0 DPN CONCENTRATION

(M/L x IO41

FIG. 2

w--- / 0

/ /

cl

? / t I I

9.0 IO.0 Il.0 12.0

PH FIG. 3

FIG. 2. The effect of DPN concentration. The details of Fig. 1 are applicable here except that the hydroxylamine concentration was maintained at 1.34 M and varying amounts of DPN were added to give the indicated final concentrations.

FIG. 3. The effect of pH. 0.033 M sodium pyrophosphate and 0.27 M hydroxylamine were adjusted to the indicated pH with hydrochloric acid or sodium hydroxide, and DPN was added to a final concentration of 1.67 X lo+ M. The change in optical density at 315 rnp was recorded.

the concentrations of the various reactants and products in so far as was possible. This will be discussed below.

2 The extent of the reaction between DPN and hydroxylamine is measured by the increase in optical density at 315 rnp due to the formation of DPN-NHOH. By analogy with the other DPN addition compounds, i.e. DPNH (16, 17), DPN-CN (6), and DPN-dihydroxyacetone (S), and by comparison with the enzyme-catalyzed formation of a DPN-hydroxylamine complex (3), the extinction coefficient of the DPN-NHOH has been assumed to approximate 6 X lo6 sq. cm. per mole, and all concentrations of DPN-NHOH have been calculated on this basis. The term equilibrium position refers to the amount of DPN-NHOH formed under the conditions described. This is expressed as AEZIS or as micromoles.


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23fect of DPN Concentration-A constant increase in the equilibrium position of the reaction (Equation 1) was observed, as indicated in Fig. 2, by maintaining the initial hydroxylamine concentration at 1.3 M and the pH at 10.0 and varying the DPN concentration from zero to 3.3 X low4 M.

Rifect of pH-Fig. 3 illustrates the effect of varying the hydrogen ion activity upon the reaction (Equation 1). The hydroxylamine concentration was 0.27 M, and the DPN concentration was 1.67 X 10m4 M initially. The log of the equilibrium position shows a linear relationship to the final pH of the reaction solution. The slope of this line is 1 and is indicative

TABLE I

Specificity of Reactants

2.5 RI solutions of the various amines indicated were adjusted to pH 10.7 with either potassium hydroxide or hydrochloric acid, and DPN was then added to a concentration of 8.3 X 10m5 M. The change in optical density at 315 rnp was noted and the extent of DPN reacting calculated. 2.6 M hydroxylamine at pH 10.6 was allowed to react with the various pyridine derivatives (added to obtain a concentration of 8 x 10m6 M).

Amines

Pyridine derivatives

Hydroxylamine 84 Methoxyamine 82 Monoethylamine 0 Diethylamine 0 DPN 84 Nicotinamide riboside 66 Ni-Methylnicotinamide chloride 17 N-Methylnicotinic acid 0 Nicotinamide 0

E -

mxtent of reaction*

* Calculated as the percentage of possible absorption at 315 rnr due to the pyridine derivatives present (see foot-note 2).

of a direct relationship of hydrogen ions to the reaction as indicated in Equation 1. The break in the curve at high pH may be due to the relatively complete reaction of DPN. The extent of reaction at pH 12.8, calculated with an extinction coefficient of 6 X lo6 sq. cm. per mole, indicates that all of the DPN is in the form DPN-NHOH.

SpeciJicity of Reactants-Table I lists various amines and pyridine derivatives tested for ability to react under these conditions. Both hydroxylamine and methoxyamine (at 2.5 M concentration) reacted to the same extent with 8.3 X 1OV M DPN at pH 10.7. Neither monoethylamine nor diethylamine reacted with DPN. DPN and nicotinamide riboside both reacted almost equally well with hydroxylamine (2.6 M) at pH 10.6. N’- Methylnicotinamide chloride reacted with hydroxylamine at a slow rate.


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R. M. BURTON AND N. 0. KAPLAN 451

N-Methylnicotinic acid and nicotinamide did not react. This reaction, therefore, has the same pyridine ring specificity that the other chemical reactions of DPN have (4-8). The amine apparently requires an “activated” nitrogen, as both hydroxylamine and methoxyamine have an oxygen attached to the nitrogen.

Spectrum of DPN-NHOH-DPN (1.0 X lo4 M) and hydroxylamine were allowed to react at pH 10.3, and the spectrum of the product was determined from 250 to 400 rnp, with appropriate corrections for the spec-

0

WAVELENGTH (rnp)

FIG. 4. Spectrum of DPN-NHOH. 2.6 M hydroxylamine adjusted to pH 10.7 with potassium hydroxide was allowed to react with 1.0 X lo+ M DPN in a volume of 3.0 ml. The spectrum was recorded with a reference cell containing 2.6 M hydroxylamine (pH 10.7).

trum of hydroxylamine. Two maximal peaks were observed, one at 260 rnp due to absorption by the adenine moiety and one at 315 rnp due to the hydroxylamine-nicotinamide moiety configuration (see Fig. 4). This is not a “true” spectrum, since this is an equilibrium and all of the DPN under the conditions of the experiment had not reacted. Hence the 260 rnp peak is too high when compared to the peak at 315 rnp. It was observed that as the 340 rnp band increased the 260 rnp band decreased slightly. This decrease at 260 rnp is similar to that observed during DPNH formation (see also the preceding paper (3)).

Equilibrium Constant of DPN-Hydroxylamine Reaction-Table II presents a list of the equilibrium constants (K) determined for the reaction


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(Equation 1) under conditions representing various concentrations of reactants (DPN and hydroxylamine) and of one product (hydrogen ion). The average K is 2.7 X lo-“. The equilibrium constant represents the

TABLE II

Equilibrium Constant of DPN-NHOH Formation

DPN and hydroxylamine were permitted to react at the indicated hydrogen ion concentrations in 0.033 M sodium pyrophosphate. The initial concentration of hydroxylamine was always large and assumed to be unchanged by the formation of DPN-NHOH. The DPN-NHOH concentration was estimated from the change in optical density at 315 mp with an extinction coefficient of 6 X lo6 sq. cm. per mole (see foot-note 2). The initial DPN concentration was known; by subtracting the DPN-NHOH final concentration, the final DPN concentration could be determined. The pH was measured after the reaction was completed.

DPN, moles per liter X lo-’

3.31 0.027 0.027 10.0 3.01 3.30 0.035 0.067 10.0 1.57 3.22 0.118 0.133 10.0 2.57 3.12 0.223 0.267 10.0 2.67 2.73 0.615 0.667 10.0 3.38 0.61 1.06 1.34 10.0 1.30 1.31 2.03 1.34 10.0 1.16 0.34 0.50 1.34 10.0 1.10 3.13 2.08 0.170 14.8 5.78 3.21 1.34 0.170 7.60 1.86 1.40 1.94 0.170 0.632 5.14 0.52 2.82 0.170 0.10 3.19

DPN-NHOH, moles per liter

x 10’ I H+, moles per

liter X 10-I’

Average..................................... .._.. 2.74

= (DPN-NHOH)(H+) (DPN)(NHzOH) ’

x lo-”

following relationship where brackets represent the molar concentration, paremheses the activity, and f the activity coefficient.

K = [DPN-NHOHI (H+) x fDPN-NHOH

[DPNl[NHzOHl fDPNfNH%OH

The ratio of the activity coefficients involved in estimating (DPN-NHOH) and (DPN) approximates 1, since the concentrations of these reactants are very small and (DPN-NHOH) and (DPN) have been assumed to be equal to the respective molar concentrations. However, (NHZOH) probably is not equal to the molar concentration of hydroxylamine, since hydroxylamine is present in high concentrations. The value of fNIlzoH is not known and has been assumed to be 1 for these calculations; therefore, (NHzOH) has been assumed to approximate its molar concentration. That


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this assumption does not introduce too great an error is indicated by the relatively constant K. Since an error may exist, no thermodynamic calculations have been presented.

Dissociation of DPN-NHOH by Dilution of Hydroxylamine-2.2 M hydroxylamine was allowed to react with 1.0 X 1OW M DPN at a final pH 10.0, and appropriate aliquots were added to a 2.6 M hydroxylamine solution at pH 10.0 and to a 0.033 sodium pyrophosphate solution at pH 10.0. The optical density (315 mp) of each solution was determined and corrected for absorption by hydroxylamine or sodium pyrophosphate. The final concentration of DPN was 7.5 X lop5 M in each dilution. The final pH was 10.0, and the concentration of hydroxylamine represents a lo-fold dilution, being 2.6 M in one instance and 0.26 M in the other dilution. The DPN- NHOH concentration changes from 4.5 X 10e5 to 0.62 X 1O-5 M, representing a decrease in the DPN-NHOH concentration of 86 per cent. The addition of ethanol to a final concentration of 1.0 M and yeast alcohol dehydrogenase showed a recovery of 6.4 X lop5 M DPN in the IO-fold diluted aliquot.3 The amount of free DPN present plus the DPN involved in the DPN-NHOH compound accounts for 93 per cent of the DPN added initially.

Dissociation of DPN-NHOH in Presence of Yeast Alcohol Dehydrogenase- DPN-NHOH was formed from 2.2 M hydroxylamine and 1.0 X 1O-3 M

DPN at pH 10.0. Aliquots mere added to (1) 2.6 M hydroxylamine, (2) 2.6 M hydroxylamine plus 1.0 M ethanol, and (3) 2.6 M hydroxylamine plus 1.0 M ethanol and 40 y of yeast alcohol dehydrogenase. Fig. 5 records the spectra with the appropriate solvent and reagent corrections. The spectra for aliquots (1) and (2) were the same, suggesting that ethanol does not displace hydroxylamine from DPN-NHOH. The spectrum of (3) after 50 minutes shows a displacement of the 315 rnp peak to 340 mp.3 This is as expected for the oxidation of ethanol by DPN and alcohol dehydrogenase when the equilibrium of the reaction (Equation 1) is displaced to the left with a reduction of the DPN-NHOH concentration and loss of 315 rnp absorption and a concurrent increase in absorption at 340 rnp due to DPNH formation. The absorption at 340 rnp is not sufficient to ac- count for the complete conversion to DPNH. The slight lack of symme- try of the 340 rnp band indicates some remaining DPN-NHOH and sug- gests an over-all equilibrium between Equation 1 and the alcohol dehydrogenase reaction (Equation 2).

Ethanol + DPN+ @ acetaldehyde + DPNH + H+ (2)

3 While hydroxylamine is an inhibitor of alcohol dehydrogenase, this inhibit,ion is competitive and high concentrations of ethanol can overcome the hydroxylamine effect. Even at these concentrations (2.6 M hydroxylamine, 1.0 M ethanol), considerable inhibition of the reaction rates is noted.


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Dissociation of DPN-NHOH by Increased Hydrogen Ion Concentration- The addition of hydrogen ions to a solution satisfying the reaction (Equa- tion 1) at equilibrium should disturb the equilibrium and shift the reac-

WAVELENGTH (mp)

FIG. 5. The dissociation of DPN-NHOH and the formation of DPNH in the presence of alcohol dehydrogenase. 2.2 M hydroxylamine was allowed to react with 1 X 10-s M DPN at pH 10.0. The spectra of aliquots were recorded without ethanol and alcohol dehydrogenase, with 1.0 M ethanol, and with 1.0 M ethanol and 40 y of yeast alcohol dehydrogenase. A reference cuvette contained hydroxylamine (2.2 M, pH 10.0).

TABLE III

Dissociation of DPN-NHOH by Increased Hydrogen Ion Concentration

DPN-NHOH was formed as described in the legend to Table II. Aliquots were removed and assayed in 2.5 M hydroxylamine (pH 10.5) for DPN-NHOH. The DPN-NHOH was adjusted to pH 6.2 with concentrated hydrochloric acid; aliquots were removed and assayed in 2.5 M hydroxylamine (pH 6.0) for DPN-NHOH and in 0.033 M sodium pyrophosphate-1.0 M ethanol (pH 9.3) for DPN with yeast alcohol dehydrogenase.

DPN DPN-NHOH DPN recovery

pm&s pVdP2 per cent

DPN (initially added) 1.84 DPN-NHOH (pH 10.5) 1.7

“ ( “ 6.0). _. _. _. 1.71 0.09 93

tion to the left, liberating DPN and hydroxylamine. Table III presents data which show that DPN is liberated upon lowering the pH of a solution of DPN-NHOH. DPN-NHOH was prepared at pH 10 in a small volume, as before, and the optical density of an aliquot in a 2.6 M hydroxylamine solution at pH 10.0 was measured. The pH of the DPN-NHOH solution was adjusted to pH 6.0 with hydrochloric acid, and the optical density of


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an aliquot in a 2.6 M hydroxylamine solution at pH 6.0 was determined. Aliquots were then assayed for DPN in hydroxylamine-ethanol solution3 (pH 10) and in sodium pyrophosphate-ethanol solution (pH 10) with yeast alcohol dehydrogenase. The data presented show that, at pH 10.0, 1.7 pmoles of DPN-NHOH were formed from 1.84 pmoles of DPN. After the pH was adjusted to 6.0, only 0.09 pmole of DPN-NHOH (a decrease of 92 per cent) was present and 93 per cent and 86 per cent, respectively, of the initial DPN were recovered by the assay procedures described.

_ DPN-NHOH

+ 1.00

4 6 8 IO

76 2 0

I Z I

E n

1

PH FIG. 6. Acid dissociation of DPN-NHOH and DPN-NHOCH,. DPN-NHOH

curve, details the same as those in Fig. 4 except that the pH was gradually decreased with hydrochloric acid and the optical density at 315 rnp at each lower pH noted. DPN-NHOCH, curve, obtained in an identical manner except that 0.80 X IO-4 M DPN and 2.6 M methoxyamine (pH 10.7) were used. The ordinate scale used to plot Curve 1 is at the left of the figure; the ordinate scale used to plot Curve 2, to the right. The slopes (m) are for each line.

DPN-NHOH and DPN-NHOCH3 were prepared at pH 10.25 and 10.55, respectively, as described previously. The pH of each solution was gradually decreased by the addition of hydrochloric acid. At each new pH, the change in the optical density at 315 rnp was recorded, and the log of the ratio of the DPN to DPN-NHOH or DPN-NHOCH3 present was plotted against the respective pH. These plots are presented in Fig. 6. The derivation of this type plot is obtained from the equilibrium equation for Equation 1.

K = (DPN - NHOH)(H+)

(DPN) (NHgOH) -


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Since the hydroxylamine concentration is large, any change caused by the formation of DPN-NHOH is negligible. Thus a new constant incorporat- ing the relatively unchanged hydroxylamine concentration may be written

(a) K’ = K(NHzOH) = (DPN - NHOH)(H+)

DPN)

On rearranging terms

K’ (DPN - NHOH) ~ = (H+) (DRY)

and taking the logarithm of each side of the equation

(4 K’

log (H+) = log (DPN - NHOH)

@J-W

Since pK’ = log l/K’ and pH = log l/(H+), then

(4 (DPN)

log (DPN _ NHOH) = - PH + PK’

Therefore, in a plot of log (DPN)/(DPN-NHOH) versus pH (equation (d)) the intercept where log (DPN)/(DPN-NHOH) = 0 gives pK’ = pH. The slope of the line is defined by expression (d) and is the negative value of the number of hydrogen ions involved in the reaction (Equation 1). In the case of hydroxylamine and DPN the slope should be - 1 .O, and experimentally this is found to be true. The pK’ determined experimentally is 9.6. This corresponds to K = 9.7 X lo-l1 and is to be compared to the value of K = 2.7 X lo-l1 previously mentioned in the paper. The change of the DPN-NHOH curve and the different curve obtained for the DPN-NHOCHJ dissociation are discussed later.

DISCUSSION

Gensler (18) discusses the reaction of hydroxylamine with cotarnine, a derivative of the heterocyclic compound isoquinoline. This reaction is of considerable interest, since the pyridine nucleus of DPN and TPN reacts chemically in a manner similar to that of any of the heterocyclic compounds, such as pyridinium, quinolinium, isoquinolinium, and acridinium salts. Variations in the reactivity of these compounds will, of course, exist, owing to ring substituents and the presence or absence of condensed rings in the individual compounds. The reactions of hydroxylamine with cotarnine (19-21) and with quinoline (22, 23) have been described previously. Gensler’s summary of the possible reaction mechanisms is presented in Equations 3 and 3, a (18) of the accompanying scheme.


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OCH, OCH, NHOH

I \

(31 P

COTARNINE J4 P

CH,CH,-N ‘CH,

/ CH=NOH

OCH, 0 OH

III

, f

OXIME

/ NH,OH

(3d) / /H CH,CH,-N

‘CH,

C /H

QO OCH, OH OCH,

lx P

COTARNINE PSEUDO-BASE

Reaction of cotarnine (I) with hydroxylamine and alkali leads to the formation of the oxime (III), which has been isolated (19-21). The oxime formed in the similar reaction with N-alkylquinolinium salts has been isolated (22, 23). The formation of the pseudobase of N-alkylquinoline from N-alkylquinolinium salts in alkaline solution has been demonstrated by Hantzsch and Kalb (24) who reported a decrease in electrical conductivity and in alkalinity of an alkaline solution of N-alkylquinolinium salts. This is due to the loss of the charge on the heterocyclic nitrogen because of the uptake of hydroxyl ions. These workers could not show the same effect with N-alkylpyridinium salts. The formation of the aldehyde tautomer (V) of the pseudobase (IV) was indicated to these workers by the formation of the oxime and phenylhydrazone of N-alkylquinolinium salts (22, 23) and the oxime of cotarnine (19-21). Equation 3, a presents the reaction sequence that would describe the oxime formation of cotarnine if the carbinol-amine-aldehyde tautomerism was involved. Thus, cotarnine (I) is in equilibrium with the carbinol-amine form (IV), which is in equilibrium with its tautomer (V), an aldehyde that can form the oxime (III). Other workers feel that this reaction can be explained without postulating


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the open ring aldehyde. Equation 3 presents the sequence involved from cotarnine (I) to a hydroxylamine addition compound (II) that can rear- range to the oxime (III).

The reaction of hydroxylamine with DPN seems to be best explained by a mechanism similar to Equation 3 except that the reaction may stop at the hydroxylamine addition step rather than proceed to the oxime form. That t,he oxime is not formed is indicated by the rapid dissociation of the DPN- NHOH both upon dilution and upon decreasing the pH of the solution, with the regeneration of enzymatically active DPN. Since it has not been possible to demonstrate the presence of the pseudobase of N-alkylpyridinium salts in mildly alkaline solutions by conductance measurements and since partitioning of the pseudobase and the quaternary salt between non- polar and polar solvents indicates that the pyridinium form is very stable (24), it seems unlikely that the rate of reaction of hydroxylamine and DPN would be so rapid (< 5 seconds) as to attain equilibrium if the pseudobase and aldehyde tautomer were involved. Also, for the above reason, the possible reaction sequence involving the elimination of water between the pseudobase and hydroxylamine seems equally unlikely.4 It is suggested below that the hydroxylamine adds to the pyridine ring at position 4; this has, however, not been proved. If this can be shown to be the point of attachment, then the possibility of involvement of the aldehyde-tautomer of the pseudobase would be definitely excluded.

Burton and Kaplan (8) suggested a mechanism for the reaction of pyridine nucleotides and certain pyridine derivatives with ketones. This reaction mechanism may be generalized in three steps: (1) The pyridine derivative exists in solution as a resonating structure with a positive charge distributed between the 1, 2, 4, and 6 positions and may be written with structures as indicated in Equation 4. The 4 position appears to

R R R

4 It has been observed that the production of the fluorescent product of DPN by alkali proceeds through at least two steps (4). First, a product is formed in the presence of alkali which has a spectral peak at 340 rnp (thought to be due to pseudobase formation) ; second, the first product is changed in the presence of alkali to give a fluorescent product having a spectral peak at 360 rnp. Unpublished results of Dr. L. Astrachan, of this laboratory, show that the addition of hydroxylamine to the first product mentioned above (340 rnp peak) inhibits the second reaction; i.e., the formation of a 360 rnp peak and the appearance of a fluorescent product. Further, a product with a peak at 325 to 330 rnp is formed. The result,s might be interpreted as following the sequence formulated in Equation 3, a of the text.


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carry the greatest positive charge, as indicated by Pullman and coworkers (25) for the enzymatic and chemical reduction of DPN and by Rafter and Colowick (26) for the chemical reduction of W-methylnicotinamide salts. (2) The other reagent, i.e. ketones, hydroxylamine, cyanide, etc., loses a proton to become a negatively charged ion. (3) The nucleophilic reagent combines ionically with the positively charged carbon 4 of the pyridine derivative, forming the reduced compound.

Hydroxylamine will form metal salts of the type MeONH2 with sodium, calcium, and zinc (27). Hydroxylamine will form oximes in basic solutions, presumably by forming an anion (Equation 5 and 5, a) that can then add to aldehydes and ketones (10-14).

NH*OH + OH*RHOH + HOH (5)

NHOH*NHzO (5, d

That the negatively charged nitrogen form is the attacking species may be inferred from the various hydroxylamine reactions. Oxime formation involves a nucleophilic attack upon a positively charged carbon atom forming a C-N bond. The action of hydroxylamine and potassium hydroxide on certain heterocyclic compounds yields amino derivatives (28). The possibility is not excluded, however, that the oxygen forms the initial bond, with a subsequent rapid rearrangement to a C-N bond. Step (3) of the DPN-hydroxylamine reaction could be formulated as in Equation 6 (other possible formulations are obvious and are not excluded). Equation 1 formulates the over-all hydroxylamine-DPN reaction; i.e., the sum of the reactions formulated in Equations 4, 5, and 6. The evidence presented in ‘:

ce”,~z + :HOH zgzfz (6 1

RI RI

this paper is consistent with such a reaction mechanism. The necessity of DPN, hydroxylamine, and a low hydrogen ion concentration has been shown. The freely reversible nature of the reaction has been demonstrated by the calculation of a constant, K, with varying concentrations of reactants. The DPN-NHOH has been shown to be dissociable by reducing the hydroxylamine concentration and by increasing the hydrogen ion concentration.

The influence of the hydrogen ion concentration on the dissociation of the compound is illustrated in Fig. 6. The curve is found to obey the ex-


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pression derived from Equation 1 over a large change in DPN-NHOH concentrations (with a change from pH 10.25 to 9.5). However, at pH 9.4 a break in the curve occurs and a new straight line with a slope of -0.1 is obtained. The only effect of increased hydrogen ions should be to increase the extent of dissociation by a direct effect upon the reaction (as depicted in Equation 1) or by the less direct effect of removing the reactive form of hydroxylamine (see Equation 5 and 5, a) from solution. However, the curve with a slope of -0.1 represents a decrease in the extent of dissociation of the DPN derivative. This possibly indicates that another DPN- compound has been formed: one that dissociates to a lesser extent than does DPN-NHOH in respect to decreased pH. The identity of this DPN compound is unknown. The curve representing the dissociation of DPN- NHOCHZ, may be interpreted in a similar manner. The line with its slope5 of -2.1 indicates that either 2 hydrogen ions are involved in the reaction or that 1 hydrogen ion is involved, as would be expected from the reaction analogous to Equation 1, and in addition the methoxyamine gains a proton much more easily than does hydroxylamine and that the methoxyamine anion is being removed from solution and thereby causing a more rapid dissociation of DPN-NHOCH, than of DPN-NHOH for the same pH inter- vals. The line of slope equal to -0.12 could be explained by the decomposition of DPN-NHOCH3, as was suggested for the DPN-NHOH curve of slope = -0.1.

Kaplan and Ciotti (1) described the competitive inhibition of liver and yeast alcohol dehydrogenase by hydroxylamine. Preincubating the liver enzyme with DPN and hydroxylamine results in the formation of an inactive compound (2). This inactive compound blocks the site of reaction on the enzyme, slowing (reversibly) the rate of the oxidation of ethanol, and is therefore a competitive inhibitor. The DPN and hydroxylamine concentrations required for half maximal inhibition are the same; i.e., 1 X 1OW and 2.5 X 1OW M, respectively. The formation of the inactive compound as a DPN-hydroxylamine compound was directly demonstrated by Kaplan and Ciotti (3), using large amounts of crystalline horse liver alcohol dehydrogenase. In this last paper (3) a discussion of the spectral shift of the DPN-hydroxylamine compound and the enzymatic DPN-hydroxylamine compound was presented and compared to a similar spectral shift observed with free and enzyme-bound reduced DPN. These results indicate that the alcohol dehydrogenase binds and activates hydroxylamine and DPN and that these activated reagents then react to form the inactive compound (2, 3). In a similar manner, a hypothesis may be advanced that the enzyme binds and activates DPN and ethanol and that t,hese activated

5 It should be noted that the two curves in Fig. 6 are drawn with different scales along the ordinate.


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reagents react to form an intermediary compound that can be decomposed into the reaction products, DPNH and acetaldehyde. This has been dia-

H HO,C=

H* CH3 REDUCED

SUBSTRATE

I1

H o=c’

OXIDIZED3

“0-g H* CH3

ticoN:;;

> c

VFlA2 > yjt~;~~2~~~oNH~

Y N

RI A, R TRANiITION

k OXIDIZED ADDITION REDUCED

COENZYM E COMPOUND STATE COENZY ME

FIG. 7. A hypothetical mechanism for alcohol dehydrogenase action. The details are described in the text. The pyridine nucleus is drawn in a stereospecific manner to indicate compliance with recent deuterium studies (29-31). The asterisk indicates a compatibility of this mechanism with the recent demonstration of a direct, stereospecific hydrogen transfer from substrate to DPN (29-31). Enzyme binding of the substrate and coenzyme (not shown in the scheme) is necessary for realization of the above considerations.

FIG. 8. Relative molecular dimensions of ethanol and hydroxylamine. The atomic radii used are from Leermakers and Weissberger (32).

grammed in Fig. 7. RI is adenosine diphosphate ribose. This scheme allows the interpretation of the competitive (with ethanol) inhibition of alcohol dehydrogenase by hydroxylamine. In Fig. 8 the molecular dimensions of ethanol and hydroxylamine are compared. The parts of the


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molecules that are involved in the reaction with DPN are of a very similar size and shape, whether hydroxylamine is considered to react with a negative charge on the nitrogen or on the oxygen (Equation 5, a). Fig. 7 represents the reaction as occurring in the following steps. (1) The reduced substrate ionizes under the influence of the enzyme and combines with the oxidized coenzyme to form an addition compound. This may be the same as the chemical addition derivative of DPN in the case of hydroxylamine. Investigations are under way in this laboratory in an effort to demonstrate a chemical reaction between DPN and ethanol. (2) The enzyme-bound addition compound could then form a transition state intermediate,6 such as is postulated in Fig. 7. (3) The transition compound decomposes into reduced coenzyme and oxidized substrate. The reaction products can then dissociate from the enzyme surface. A competitive inhibitor, such as hydroxylamine, will be enzyme-bound and activated and will form an addition compound. This addition compound is not able to form a transition intermediate, as illustrated, and to decompose to give reaction products. The firmly bound inactive compound, occupying the activating enzyme sites, prevents the normal reaction from occurring. All of the steps are written as reversible, since the gross reaction is reversible and since the inhibition by hydroxylamine is competitive. This scheme is consistent with the work of Fisher et al. (29), Loewus et al. (30), and Vennesland and Westheimer (31) which showed a direct hydrogen transfer from the substrate to the coenzyme for both the yeast and liver’ alcohol dehydrogenase systems. A similar enzymatic reaction mechanism has been discussed by Snell (33) in an effort to explain the action of transaminases requiring pyridoxal phosphate. A non-enzymatic transamination reaction between the coenzyme and substrates has been demonstrated.

The comments and criticisms of Professor W. Mansfield Clark are grate- fully acknowledged. Numerous discussions of the chemical reaction mechanism with Dr. A. San Pietro and Mr. S. Kinsky are appreciated. We wish to thank Mr. Francis Stolzenbach for his assistance on parts of this work.

SUMMARY

1. A chemical reaction is described that requires diphosphopyridine nucleotide, hydroxylamine, and hydroxyl ions. The product of this reaction has an absorption maximum at 315 rnp and at 260 rnp. As the 315 rnp band is forming, a concurrent decrease in the 260 rn,u band is observed. The product is readily dissociable, regenerating free (enzymatically active)

6 The use of the term transition state intermediate is not intended to exclude other transition states that may be required for the complete reaction.

7 Personal communication, Dr. B. Vennesland.


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diphosphopyridine nucleotide. An apparent equilibrium constant has been calculated; K = 2.7 X IO-” for the reaction, i.e. diphosphopyridine nucleotide plus hydroxylamine to yield the product and hydrogen ions.

2. A possible reaction mechanism is present and discussed. 3. The relationship of diphosphopyridine nucleotide addition products

to a possible enzymatic mechanism of liver alcohol dehydrogenase action is presented and discussed.

BIBLIOGRAPHY

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Robert Main Burton and Nathan O. KaplanDIPHOSPHOPYRIDINE NUCLEOTIDE

HYDROXYLAMINE WITH A CHEMICAL REACTION OF

1954, 211:447-463.J. Biol. Chem.

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A CHEMICAL REACTION OF HYDROXYLAMINE WITH DIPHOSPHOPYRIDINE NUCLEOTIDE