Enzymic properties of chemically prepared diphosphopyridine nucleotide analogs

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BIOCHEMISTRY

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BIOPHYSICS

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107-120 (1957)

Enzymic Properties of Chemically Prepared Diphosphopyridine Nucleotide Analogs’ Robert Main Burton2 and Nathan 0. Kaplan From the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland Received July 26, 1956

The preceding paper (1) describes the preparation of pyridine nucleotide analogs by the addition reaction of pyridine nucleotides with carbony1 compounds followed by potassium ferricyanide oxidation of the addition product. This paper presents the results of studies comparing the biochemical properties of diphosphopyridine nucleotide (DPN) analogs formed from DPN-dihydroxyacetone,3 DPN-glyceraldehyde, and 1 Contribution No. 127 of the McCollum-Pratt Institute, The Johns Hopkins University. Aided by grants from the Rockefeller Foundation, the American Cancer Society as recommended by the Committee on Growth of the National Research Council, and the National Cancer Institute, the National Institutes of Health, Grant iy C-2374- (c). 2 Research Fellow of the National Heart Institute, U. S. Public Health Service. Present address: The National Institute of Neurological Diseases and Blindness, National Institutes of Health, Bethesda 14, Maryland. 8 The following designations will be employed in this paper: ii ii a. ribose or ribosyl -0-P-0-P-0-ribosyladenine, I I oob. isonicotinic acid hydrazide, INH; c.

ADPR or ADPR-;

Oxidized form or analog Rl I\_ / I

Reduced form or addition compound H RI -CONHz

CONHs II

I

I

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BURTON AND KAPLAN

DPN-acetone, in the pig brain, beef spleen, and Neurospora crassa DPNase systems. The products of the DPNase reactions are described and discussed. EXPERIMENTAL The materials and methods used are in general the same as those described in the precedingpaper. The procedures of Zatmanet al. wereused for the preparation of pig brain DPNase (2) and beef spleen DPNase (3). Neurospora crassa DPNase was prepared by the procedure of Kaplan et al. (4). Crystalline yeast alcohol dehydrogenase was prepared by the procedure of Racker (5), lactic acid dehydrogenase by the method of Kornberg and Price (6), crystalline 3-phosphoglyceraldehyde dehydrogenase according to Cori, Slein, and Cori (7), glycerol dehydrogenase by the method described by Burton (S), and reduced pyridine nucleotide oxidase from Clostridium kluyveri by the method of Weber and Kaplan (9).

Hydrolysis

of DPN-D,,(I)

and DPN-D,,(II)

Mixtures

by DPNase

One gram of DPN was used to prepare the oxidized form of DPN-dihydroxyacetone as described previously (1). Following the potassium ferricyanide oxidation of the reduced nucleotide, 60 ml. of pig brain DPNase (16 units/ml.) was added, and the reaction mixture was incubated for 20 hr. at 37°C. The mixture was centrifuged for 30 min. at high speed in a Servall centrifuge, and the supernatant liquid was passed over a Dowex I-formate column to separate the components as described under Results.

Isolation of ff The (r fractions from several Dowex 1 column separations of DPNase-hydromixtures were combined and passed through lysed DPN-D,,(I) and DPN-D,,(II) a fresh Dowex 1-formate column to insure removal of the nucleotides and iron salts. The solution was made 0.5 N with respect to hydrochloric acid, and the resulting gelatinous protein precipitate was removed by filtration. The filtrate was (1) Ri = H Rz = ADPR-

DPN or diphosphopyridine nucleotide DPN-D,,(I) and DPN-D,,(D) DPN-G,,(I) and DPN-G&II) DPN-A,,(I)

DPNH

(2) R1 = dihydroxyacetone moiety DPN-D or DPN-dihydroxyacetone R.J = ADPRDPN-G or (3) Ri = glyceraldehyde moiety DPN-glyceraldehyde Rz = ADPRDPN-A or (4) Ri = acetone moiety DPN-acetone Rz = ADPRN-I Ni-methyl (5) RI = acetone moiety nicotinamide-acetone Rz = CHs The reman numeral following the abbreviation indicates the net negative charge on the nucleotide at pH 7 as determined by paper electrophoresis. The possible structures of the dihydroxy, glyceraldehyde, and acetone moieties are presented in Eq. (5) of the paper by Burton et al. (1).

ENZYMIC

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OF DPN ANALOGS

109

taken to dryness under vacuum. The yellow-white residue was dissolved in 10% aqueous alcohol and then precipitated with an excess of alcohol. The precipitate was ether-washed and air-dried. The compound is a yellow waxlike solid that fluoresces yellow. The uncorrected melting point is 217-222’C., with darkening occurring at 183°C. Alpha is water-soluble and ether-insoluble. It gives a benzidine test for nitrogen, a white precipitate with silver nitrate, and a positive ferric chloride test. It does not decolorize bromine in carbon tetrachloride, but does decolorize bromine water. Alpha gives a positive Holman test for aromatic amides. The ultraviolet absorption spectrum of a shows a 230-rnN peak in acid solution.

Isolation of /3 The combined fl fractions in 1 N formic acid were reduced to a small volume under vacuum to yield 20 ml. of a gold-colored solution. This was extracted ten times with equal volumes of ether, until neither the aqueous nor ether phase tested acid with Hydrion paper. The aqueous phase was reduced in volume under vacuum, and six times thevolumeof absoluteethanol was added. The brown gelatinous precipitate was removed. The filtrate was gold in color with a bright gold fluorescence. The filtrate was taken to dryness under vacuum; the residual brown tar was dissolved in a little water, and the volume was reduced to 1 ml. Adding 15 ml. of absolute ethanol yielded a flocculent precipitate that was ether-washed. This hygroscopic black tar was dissolved in 0.5 ml. of water and precipitated with 1 ml. of ethanol. The precipitate was ether-washed and dried over phosphorus pentoxide to yield a light-brown solid. The uncorrected melting point was greater than 315°C. with darkening at 230°C. This compound, B, is water-soluble and ether-insoluble; it gives a positive ferric chloride test, decolorizes potassium permanganate and bromine water, and gives a questionable silver nitrate test. It gives a positive test for an aromatic amide group. The spectra of 6 show a 260-rnr peak in acid and alkali. RESULTS

Enzymic Hydrolysis of the Analogs The data presented in Table I show the enzymic hydrolysis of DPND,(I) and DPN-D&II) by pig brain and beef spleen DPNases but not by Neurospora DPNase. It may be seen that DPN is hydrolyzed as expected yielding ADPR and nicotinamide; DPN-D,(I) and DPN-D,,(II) are hydrolyzed and show spots on the chromatogram with Rf values corresponding to ADPR and, presumably, the substituted nicotinamide moieties of DPN-D,,(I) and DPN-D&II) (designated (Y and 8; see below). These latter spots fluoresce under ultraviolet light. Spots with R, values corresponding to those of the dinucleotides are absent, indicating complete cleavage of the pyridinium ribosidic link. Hydrolysis of DPN-D,(I) and DPN-D&II) by beef spleen DPNase was incomplete, and the dinucleotide spots were observed along with the ADPR, nico-

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TABLE I of DPN, DPN-D,,(I), DPN-D,(II)

Hydrolysis

and DPN-A,,(I)

by

Nucleotidases One milligram of the nucleotide was incubated with 0.2 ml. of pig brain DPNase (16 units/ml.) in 0.25 ml. potassium phosphate buffer (0.2 ilf; pH 7.6) for 2 hr. at 37°C. The reaction mixture was centrifuged for 45 min. and an aliquot of the supernatant liquid was chromatographed. The aliquots were chromatographed on Whatman #4 paper using the solvent system of Zatman et al. (12), i.e., 0.1 N acetic acid-ethanol 1: 1, for 16 hr. at 25°C. The compounds were located by their property of quenching ultraviolet light (2537 A.), or of fluorescing with ultraviolet light (3700 A.). Similar experiments were run with beef spleen or Neurospora crassa DPNase replacing the pig brain enzyme. Controls were run in which the nuoleotides were chromatoaranhed before incubation. Diphosphopyridine

Nucleotide

I

-*

Control

.Rf I. DPN

0.46

11. DPN-D,(I) III.

DPN-D,,

0.42 (II)

IV. DPN-A,(I)

0.43

=

Pig brain DPNase -~ RI -

Beef spleen DPNase

Arwros~ora C,&LW DPNase

Rf -

RJ -

0.57 0.81 0.58 0.65 0.60 0.67

0.62 0.83 0.45 0.62 0.71 0.45 0.62 0.71

0.62 0.83 0.45 0.45 0.45 0.61 0.77

-

0.45

-

Compound corresponding to obs. Rf

ADPR Nicotinamide ADPR a

ADPR B ADPR Nicotinamide-acetone moiety

-

tinamide, and the fluorescent substituted nicotinamide moieties, (Y and 8. Similar experiments have shown that DPN-G,,(I), DPN-G&II), and DPN-A,,(I) are hydrolyzed by pig brain and beef spleen DPNases. The Neurospora DPNase will not hydrolyze DPN-D,,(I), DPN-D,(D), DPN-G,(I), or DPN-G,,(H), but chromatographic evidence indicates that DPN-A,,(I) may be cleaved, but at a very much slower rate than for DPN. Enzymic Formation of DPN from the Analogs Pig brain DPNase has been shown to have transglycosidase activity, transferring the ADP-ribosyl grouping from a donor (as DPN) to a pyridine acceptor (as INH) (2). If, as has been discussed previously, the

ENZYMIC

PROPERTIES

OF

DPN

111

ANALOGS

TIME IN HOURS FIG. 1. Nicotinamide exchange: the formation of enzymically active DPN from DPN-D,,(I) catalyzed by pig brain DPNase. Five micromoles of oxidized DPN-D,,(I) were incubated with 300 pmoles of nicotinamide in potassium phosphate buffer (0.03 M; pH 7.6) in the presence and in the absence of 6 units of pig brain DPNase (16 units/ml.). The total volume was 1.3 ml.; 37°C. Aliquots of 0.1 ml. were removed and assayed for DPN in the 0.1 M glycine-O.5 M ethanol402 M nicotinamide system of Zatman et al. (3) with crystalline yeast alcohol dehydrogenase.

analogs DPN-D,,(I), DPN-D,,(D), DPN-G,,(I), DPN-G,,(D), and DPN-A,,(I) are modified only on the nicotinamide moiety, then it should be possible to transfer the ADP-ribosyl grouping from each analog to nicotinamide, in the presence of pig brain DPNase, forming enzymically active DPN. Figure 1 presents a plot of data showing the formation of DPN from DPN-D,,(I) assayed with yeast alcohol dehydrogenase and ethanol. Similar experiments with the other analogs, DPN-D,,(II), DPN-G,,(I), DPN-G&II), and DPN-A,,(I) all showed the production of enzymically active DPN by this exchange reaction. The analogs were converted to DPN in yields varying from 20 to 35 %, the remainder of the analogs being hydrolyzed to the free pyridine derivatives. Products

of the DPNase

Hydrolysis

of DPN-D,,(I)

and DPN-D,,(II)

A mixture of DPN-D,,(I) and DPN-D,,(D) was hydrolyzed with pig brain DPNase, and the cleavage products were separated by means of a Dowex l-formate coiumn. The light absorption of the eluate fractions

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from the column was determined at 260 and 390 rnp. The first peak, absorbing both at 260 and 390 rnp, represents that material that is not bound on a Dowex 1 column. These fractions were collected and combined and labeled (Y.The next peak, eluted with 0.1 N formic acid, varied quantitatively from experiment to experiment and represents unhydrolyzed nucleotide analog as indicated by paper chromatography. The third peak, possessing high 260-rnp absorption, is probably ADPR as indicated by paper chromatography. The fourth peak, possessing both 260- and 390-rnp absorption, was collected and combined as indicated and labeled B. As discussed in the preceding paper, DPN-D,,(I) probably has its dihydroxyacetone side chain intact, while DPN-D&II) probably has the side chain oxidized so that it possess a carboxylic acid grouping. Thus, the DPNase cleavage products of DPN-D,,(I) should be ADPR and a dihydroxyacetone-nicotinamide moiety. The dihydroxyacetonenicotinamide moiety (in a manner similar to nicotinamide) would be uncharged at this pH and would not be held by an anion-exchange resin. DPN-D,,( II), on hydrolysis, should give a substituted nicotinamide moiety possessing a carboxylic acid grouping and, therefore, a negative charge at pH 7.8, and would be bound by the Dowex 1 column. Thus, CYwould represent the substituted nicotinamide moiety of DPN-D,,(T), and /3 would be the moiety of DPN-D,,(D). The relationship of LYand p to nicotinic acid and nitotinamide will be shown and discussed later. Amide Test The Holman amide test (10) is essentially a Hofmann degradation of an aromatic amide forming an aromatic amine which is diazotized and coupled with (N-1-naphthyl)ethylenediamine to give a colored compound. This test gives an orange-red solution with nicotinamide. Pyridine, nicotinic acid, and nicotinyl diethylamide do not form colored derivatives. Alpha gives a positive test producing a fuchsin color showing a spectral peak near 545 rnp; 8, also giving a positive test, produces an orange-red color similar to that of nicotinamide. The colors formed by both nicotinamide and fl have peaks at 500 rnp. DPN gives a positive Holman test with a peak at 500 rnp, but with an extinction coefficient of about 55 % that of nicotinamide (11). DPN-D,,(I), DPN-D,,(D), DPN-G,,,(I), DPN-G,,(D), and DPN-A,,(I) give a positive test. The color with DPN-&,(I) is very slight, however, giving a peak absorption at 500 rnM of about 6 % that produced by an equivalent amount of DPN.

EXZYMIC

PROPERTIES

OF

DPN

113

ANALOGS

TABLE II Eflect of Chemical and Enzymic Hydrolysis of DPN and DPN-A,,(I) on the Holman Amide Test DPN and DPN-A,,(I) (1 rmole) were incubated for 30 min. at either 25” or 100°C. in potassium phosphate at pH 7.7 or at 37°C. with potassium phosphate (pH 7.7) and 30 units of beef spleen DPNase. The total volume was 4.0 ml. Following incubation, the reaction mixtures were cooled to 25°C. and analyzed for the amide grouping by the Holman procedure (10).

1OOT. in

Untreated I

phosphatebuikr

“g&S&,“,

DPN DPN-A,,(I)

Since nicotinamide produces a color with a higher extinction coefficient than DPN, then hydrolysis of DPN at the nicotinamide-ribose bond, by chemical or enzymic means, should show an increased absorption at 500 rnp from a DPN aliquot. Table II presents data showing the results of hydrolyzing DPN and DPN-A&I). DPN hydrolyzed by boiling at pH 7.7 in phosphate buffer for 30 min. or incubated with beef spleen DPNase at 37°C. showed an increased absorption at 500 rnp following the Holman test of 232 and 217 %, respectively. DPN-A,(I), before hydrolysis, had an optical density of 0.045 at 500 rnp at 10m3M. DPN&,(I) heated at 100°C. in phosphate at pH 7.7 for 30 min. now gave a Holman test color with an optical density of 0.488. However, the enzymic hydrolysis of DPN-&,(I) with beef spleen DPNase gave an optical density of 0.123 for the Holman test. The low value could be due to incomplete cleavage of DPN-&,(I) by the enzyme; however, a high enzyme concentration was employed and the hydrolysis of DPN-&,(I) should have been complete. Then, the threefold increase in the color intensity development with the Holman procedure due to splitting DPN-&,(I) by the DPNase and yielding the free nicotinamideacetone moiety would be comparable with the increase in color intensity caused by hydrolyzing DPN yielding free nicotinamide. In contrast, the chemical hydrolysis of DPN causes a twofold increase in the color intensity, but the color developed with DPN-A,,,(I) is increased tenfold by hydrolysis. This is suggestive of cyclization between the carbonyl group of the side chain of the analogs and the amide nitrogen as discussed in the

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KAPLAIG

previous paper (1). The interpretation would be that the conditions employed for the chemical hydrolysis would open the carbonyl-amide nitrogen bond in addition to cleaving the nicotinamide-riboside link, whereas the enzymic hydrolysis would affect only the nicotinamide-riboside bond. Ionophoresis of Q!and /3 Ionophoresis of (Y, 8, nicotinic acid, and nicotinamide was carried out as described in the previous paper for the nucleotides DPN-D,,(I) and DPN-D,,(D). Alpha shows the same charge relationship to nicotinamide that DPN-D,,(I) shows to DPN. Similarly, /3 shows the same charge relationship to nicotinic acid that DPN-D&II) shows to reduced DPN. Figure 2 shows that at pH 2.65 the four compounds, nicotinic acid, nicotinamide, cr, and 8, migrate together, moving slightly toward the cathode. At this pH the ionization of the carboxylic acid group is suppressed, and the pyridine nitrogen is combined with the acid forming a quaternary nitrogen which carries a positive charge, hence the movement toward the cathode. At pH 4.55 the pyridine nitrogen is still partially quaternary, but the carboxylic acid group is now partially ionized and nicotinic acid and /3 migrate toward the anode. Nicotinamide and cy show a slight migration toward the cathode. At pH 7.32, where the quaternary nitro-

CATHODE AMIDE j3 ACID o(

pH 2.65

AMIDE /3 ACID 0~.

pH 4.55

AMIDE 4CID o(

j3

pH732

ANODE 2. paper (1). fl indicate represent represent FIG.

Ionophoresis of Q and 8. The details are presented in the previous Acid indicates nicotinic acid, amide indicates nicotinamide, and (Y and the nicotinamide-dihydroxyacetone moieties. The crosshatched areas ultraviolet light (2537 A.) quenching spots, and the unshaded areas ultraviolet light (3700 A.) fluorescent spots.

ENZYMIC

PROPERTIES

115

OF DPN ANALOGS

gen formation is suppressed, a and nicotinamide do not move. The movement of /3 and nicotinic acid is toward the anode and is probably governed by the ionization of a carboxylic acid grouping. Analog Inhibition

of DPNa.se

Table III presents data that show that the analogs are able to inhibit the DPNases from pig brain, beef spleen, and Neurospora. The inhibition of DPN splitting by the pig brain enzyme is slight and is probably competitive since this enzyme readily hydrolyzes the nucleotide analogs. The beef spleen DPNase is markedly inhibited. This inhibition will be discussed further below. The Neurospora enzyme is inhibited most by DPNA,,(I), which is the only analog that is split by it. Further studies of the inhibition of DPN-A,,(I) are presented in the data of Table IV. The first experiment shows the noncompetitive inhibition of Neurospora DPNase by DPN-&,(I). At the three DPN concentrations employed, a constant percentage of inhibition is obtained. The second experiment, with beef spleen DPNase, shows that at a low concentration of DPN-A,,(I) the inhibition of the enzyme is competitive and may be reduced by the addition of more substrate. At a higher level of DPN-A,,(I), the inhibition appears to be uncompetitive, i.e., as the concentration of the DPN is increased the inhibition of the enzyme becomes greater. We have also found that the INH analog of DPN inhibits the spleen DPNase competitively, whereas uncompetitive inhibition of beef spleen DPNase has been TABLE III crassa, Beef Spleen and Pig Brain Diphosphopyridine Nucleotidases The concentrations of nucleotides employed are indicated in the table. The enzymes were employed in such concentrations that 60-70yo of the DPN was hydrolyzed in 15 min. at 37°C. without inhibitors. The DPN present (1.4 X 10-s M) was assayed by the cyanide procedure of Colowick et al. (16) which is not affected by the presence of these analogs. The Neurospora DPNase experiments were run in 0.1 M potassium dihydrogen phosphate buffer. The beef spleen and pig brain DPNase were run in 0.1 M potassium phosphate buffer (pH 7.7). Inhibition

of Neurospora

Per cent inhibition of DPNase NEWOS&WC3 Beef spleen Pig brain

1.4 x 10-s M DPN-D,,(I) DPN-D&II) DPN-G,,(I) DPN-A&I)

24 18 46 66

80 52 81 82

18 10 30 23

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TABLE Effect of DPN

Concentration

The details of Table III apply centration was varied as noted, concentrations. and two different ployed (beef spleen Iy 1 and #a).

IV

on DPN-A,,(I)

Inhibition

of DPNaaes

to these experiments except that the DPN conthe Neurospora DPNase was employed at two nrenarations of beef spleen DPNase were em- Per cent inhibition

DPNase

of DPNase

DPN concentration 2.9 X 10-J

1.4 x

lo-

(moles/l.) 0.7 x

10-s

1. Neurospora

0.2 unita 1.0 unite 2. Beef spleen Beef spleen Beef spleen Beef spleen

49

21 81 40 62 31

#la #lb #2” 12”

43 28 66 60 51 54

47 22 31 92 33 73

0 1.47 x 10-a M DPN-A,,(I). b 1.47 x 1O-4M DPN-A,,(I).

observed by Zatman et al. (12) with free INH at very low DPN concentrations. Reduced Pyridine Nucleotide Oxidase

A nonspecific reduced pyridine nucleotide oxid,ase from Clostridium Icluyveri was found to be capable of oxidizing all three of the addition compounds of DPN, i.e., DPN-dihydroxyacetone, DPN-glyceraldehyde, and DPN-acetone. The change in the spectra of these compounds (see the preceding paper) was used as the index of oxidation. The rate of oxidation of the analogs by the Cl. kluyveri enzyme was about x00 of the rate for reduced DPN. Dehydrogenases

The analogs, both oxidized and reduced forms, were inactive either as coenzymesor as inhibitors in a number of dehydrogenase systems studied, i.e., yeast alcohol dehydrogenase, beef heart lactic acid dehydrogenase, 3-phosphoglyceraldehyde dehydrogenase from muscle, and Aerobacter glycerol dehydrogenase.

ENZYMIC

PROPERTIES

OF

DPN

ANALOGS

117

Hemophilus parainfluenza requires DPN* as a nutrient and has been used as a bioassay for DPN (13). None of the oxidized analogs would support the growth of this organism; neither would these compounds inhibit the growth of H. parainJluenza on DPN. DISCUSSIOX The preceding paper described the preparation of analogs of DPN by the addition of aldehydes and ketones to the pyridine ring of DPN, followed by potassium ferricyanide oxidation of the addition compound. This treatment of DPN yields products that seem to be devoid, in general, of coenzyme or coenzyme inhibitory activities in the systems studied. However, it was found that pig brain DPNase would readily hydrolyze the DPN analogs formed from dihydroxyacetone [DPN-D,,(T) and DPN-D,,(U)], glyceraldehyde [DPN-G,,(I) and DPN-G&II)], and acetone [DPN-A,,(I)]. Beef spleen DPNase hydrolyzes the analogs, but at a rather slow rate; whereas the Neurospora DPNase will not hydrolyze the dihydroxyacetone and glyceraldehyde-DPN analogs, but will cleave the acetone-DPN analog, DPN-A,,(I). It is interesting to note that the Neuropsora DPNase is otherwise highly specific, hydrolyzing only DPN and TPN (and deamino DPN at about 2 % the DPN rate) (4) ; none of the other analogs of DPN that have been prepared are split by this enzyme, i.e., isonicotinic acid hydrazide analog of DPN (2), 3-acetylpyridine analog (15)) etc. The products of the enzymic hydrolysis of the analogs, DPN-D,,(I), DPN-D&II), DPN-G,,(I), DPN-G,, are the substituted nicotinamide moieties (II), and DPN-A,,,(I), and ADPR. The ADPR has been identified by chromatography in two solvent systems and by separation from other components on an ion-exchange column followed by paper chromatography identification. That the analogs are modified on the nicotinamide moiety is best shown by the formation of enzymically active DPN from all of the oxidized analogs. Pig brain DPN-ase possesses transglycosidase activity and can transfer the ADP-ribosyl group of DPN to any suitable acceptor (2). By incubating the various analogs of DPN with pig brain DPNase and nicotinamide as the ADP-ribosyl acceptor, ADPR-nicotinamide (DPN) is formed in yields up to 35 %. This is represented by the equation in Fig. 1. 4 H. parainjeuenza will grow on nicotinamide riboside as well as on DPN (13). Nicotinamide ribotide is equally effective as growth factor (14).

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Hydrolysis of large amounts of DPN-D,,(I) and DPN-D,,(II) with pig brain DPNase, followed by Dowex 1-formate separation of the products yielded ADPR and unsplit analogs along with the substituted nicotinamide moieties cr [from DPN-D,,(I)] and p [from DPN-D&II)]. Both a! and /3 were isolated, but in small quantities. The ultraviolet spectra of each show a peak at 260 rnp, which is typical of pyridine derivatives. Both CYand 0 give positive results with the Holman aromatic amide test. But, whereas nicotinamide quenches ultraviolet light, CYfluoresces yellow and fi fluoresces with a bright gold color. Ionophoretically, LYbehaves in a manner similar to nicotinamide. At neutral pH, neither (II nor nicotinamide migrate, since the nicotinamide molecule should be uncharged; CYis considered to possess no net charge. The tertiary ring nitrogen of nicotinamide reacts readily with acids to form a quaternary nitrogen salt. The positively charged pyridinium ion will migrate toward the cathode. This is similar to the behavior described in the preceding paper for DPN and DPN-D,,(I) in an electric field. At the pH values studied, /3 behaves in a manner close to that of nicotinic acid. Thus, in acid where the ionization of the carboxylic acid group is suppressed and the ring nitrogen has a positive charge, the pyridinium ion form of nicotinic acid will migrate toward the cathode. Beta also migrates toward the cathode, at a rate of migration similar to that of nicotinic acid, in an acid solution. In a neutral or slightly basic solution, the acid group of nicotinic acid will be ionized and the ring nitrogen will be tertiary and uncharged; therefore, nicotinic acid will migrate toward the anode. Beta is observed to migrate toward the anode under these conditions. DPN-D,,(II) was found to behave as though it possessed a net negative charge greater than that of DPN and DPN-D,,(I) as described in the preceding section. These data suggest that cy and P are both pyridine derivatives, containing amide groupings, and differ from the pyridine amides known by fluorescing instead of quenching ultraviolet light. Alpha and @ differ from each other in that p possesses a net negative charge in neutral solution while a! has a net charge of zero. DPN-D,,(I) and DPN-D&II) differ in their net charges in neutral solutions. These data are consistent with the structures postulated in the preceding section for the pyridine moieties of DPN-D,,(I) and DPN-D&II). The ketone is pictured as being attached to the pyridine ring at the 4-position through the carbon adjacent to the carbonyl group of the ketone. In DPN-D,,(I), and hence in CZ,the substituted side chain is intact; while in DPN-D&II), and thus in /3, the side chain has been pictured as being

ENZYMIC

PROPERTIES

OF

DPN

ANALOGS

119

oxidized to a carboxylic acid. The acetone derivatives, DPN-A,,,(I) and N-l, fail to give an appreciable amide test until after the compounds have been heated with mild alkali. This could be interpreted to suggest that the carbonyl group of the acetone side chain is masking the amide nitrogen perhaps by forming a second heterocyclic ring as pictured in the preceding paper. While the behavior of the analogs as inhibitors of Neurospora DPNase is typically noncompetitive, the inhibition of the beef spleen enzyme by the analog, DPN-A.,,,(I), was rather unusual. At a concentration of low4 M, DPN-&,(I) showed typical competitive inhibition of the beef spleen DPNase, and increasing the DPN to analog ratio caused a decrease in the percentage of inhibition. However, if the concentration of DPN-A,,(I) was increased to 10e3 M, inhibition occurred resembling that of uncompetitive inhibition. Thus, increasing the DPN to analog ratio resulted in an increase in the percentage inhibition. This suggests that either the observed effect is a type of inhibition different from uncompetitive inhibition or that an artifact is responsible for misleading results. Further work is needed to clarify this point. Uncompetitive inhibition of spleen DPNase by isonicotinic acid hydrazide has been observed at very low DPN concentrations by Zatman et al. (12), who explain that the substrate (DPN) is required to provide an ADP-ribosyl group to form the isonicotinic acid hydraaide analog of DPN and that the analog is the inhibitor. Thus, increasing the amount of substrate present will allow the formation of more analog which therefore causes greater inhibition. Studies of the relationship of the INH analog to DPN ratio on inhibition of beef spleen DPNase show that the INH analog inhibition is competitive with DPN. &JMMARY

Potassium ferricyanide oxidation of the addition reaction products of DPN with dihydroxyacetone, glyceraldehyde, and acetone results in the formation of oxidized analogs of the coenzyme. These analogs are hydrolyzed by pig brain and beef spleen DPNases. Only the acetone analog of DPN is hydrolyzed by the Neurospora crassa enzyme, and this occurs at a slow rate. The addition compounds are not split by any of the DPNases. The products of the DPNase hydrolysis of the analogs of DPNdihydroxyacetone [DPN-D,,(I) and DPN-D,,(II)] have been isolated. One product is adenosine diphosphate ribose. The two analogs each yield substituted nicotinamide moieties, designated (Y [from DPN-D,,(I)] and

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AND

KAPLAN

p [from DPN-D,,(D)], whose properties are presented. The synthetic analogs will inhibit the cleavage of diphosphopyridine nucleotide by the various DPNases. The Neurospora enzyme appears to be inhibited noncompetitively. The addition products of diphosphopyridine nucleotide and dihydroxyacetone, glyceraldehyde, and acetone are oxidized by the reduced pyridine nucleotide oxidase of Clostridium kluyveri at a slow rate. The analogs are not effective in any of the dehydrogenase systems studied, either as coenzyme or as inhibitors. They will not support or inhibit the growth of the DPN-requiring organism, Hemophilus parainjluenza. REFERENCES 1. BURTON, R. M., SAN PIETRO, A., AND KAPLAN, N. O., Arch. Biochem. Biophys. 70, 87 (1957). 2. ZATMAN, L. J., KAPLAN, N. O., COLOWICK, S. P., AND CIOTTI, M. M., J. Biol. Chem. 209, 467 (1954). 3. ZATMAN, L. J., KAPLAN, N. O., AND COLOWICK, S. P., J. Biol. Chem. !&IO, 197 (1953). 4. KAPLAN, N. O., COLOWICK, S. P., AND NASON, A., J. Biol. Chem. 200,197 (1953). 5. RACKER, E., J. Biol. Chem. 164, 313 (1950). 6. KORNBERO, A., AND PRICE, W. E., JR., J. Biol. Chem. 193,451 (1951). 7. CORI, G. T., SLEIN, M. W., AND CORI, C. F., J. Biol. Chem. 173, 605 (1948). in Enzymology” (S. P. Colowick and N. 0. Kaplan, 8. BURTON, R. M., “Methods eds). Academic Press, New York, 1955. 9. WEBER, M. M., AND KAPLAN, N. O., Bacterial. Proc. p. 96 (1954). 10. HOLMAN, W. M., Biochem. J. 66, 515 (1954). 11. KAPLAN, N. O., GOLDIN, A., HUMPHREY, S. R., CIOTTI, M. M., AND STOLZENBACH, F. E., J. Biol. Chem. 219, 287 (1956). 12. ZATMAN, L. J., KAPLAN, N. O., COLOWICK, S. P., AND CIOTTI, M. M., J. Biol. Chem. 209, 453 (1954). 13. GINORICH, W., AND SCHLENK, F., J. Bacterial. 47, 535 (1944). 14. BACHIJR, N. R., AND KAPLAN, N. O., Bacterial. Proc. p. 116 (May, 1955). 15. KAPLAN, N. O., AND CIOTTI, M. M., J. Am. Chem. Sot. 76,1713 (1954). 16. COLOWICK, S. P., KAPLAN, N. O., AND CIOTTI, M. M., J. Biol. Chem. 191,447 (1951).