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Studies of the enzymes involved in nicotinamide adenine dinucleotide metabolism in Aspergillus niger
BIOCHIMICA ET BIOPHYSICA ACTA
311
BBA 1233O S T U D I E S ON T H E E N Z Y M E S I N V O L V E D I N N I C O T I N A M I D E A D E N I N E D I N U C L E O T I D E M E T A B O L I S M IN
A S P E R G I L L US NIGER* D. S. R. SARMA, S. R A J A L A K S H M I AND P. S. SARMA
Department of Biochemistry, Indian Institute of Science, Bangalore (India) (Received J u n e 4th, 1963)
SUMMARY
The enzyme nicotinamide amidase (n~.cotinamide amidohydrolase) was purified 57-fold from Aspergillus niger. The purified preparation was specific towards its substrate nicotinamide and did not deamidate NADP, NAD, NMN, N'-methyl nicotinamide, asparagine, glutamine, benzamide, a-naphthaleneamide and indoleacetamide. The optimum pH was found to be 7.5. Temperature optimum was 4 o°. It had a Km value of 6.504. lO -4 M towards nicotinamide. The enzyme exhibited Mg~+ ion requirement for its optimum activity. NAD-glycohydrolase (EC 3.2.2.5) was purified Iog-fold from the mold A. niger. The enzyme preparation was active only towards NAD and NADP and did not attack NMN, N'-methylnicotinamide and NADH. The Km value for NAD was found to be 7.693. lO -6 M. The enzyme did not require any metal ion for its activity. It is suggested that A. niger will serve a better source for a large scale preparation of NAD-glycohydrolase than the Neurospora mold. The biological role of both NAD-glycohydrolase and nicotinamide amidase in the regulation of cellular NAD level has been discussed. It is, further, observed that NAD did not exert its feedback control on nicotinamide amidase at least in A. niger. INTRODUCTION
Nicotinamide amidase (nicotinamide amidohydrolase), an hydrolysing enzyme involved in NAD metabolism, was first demonstrated in lactic acid bacteria by HUGHES AND WILLIAMSON 1. Later the enzyme was found to be present in microorganisms 9-4 as well as in higher vertebrate tissues 5. However, the biological significance of this hydrolysing enzyme is yet to be understood. Only in recent years the role of nicotinamide amidase and NAD-glycohydrolase (EC 3.2.2.5) in the maintenance of cellular NAD level has been studiede, 7. According to these investigations the enzyme "nicotinamide amidase" deamidates nicotinamide to nicotinic acid and the nicotinic acid Abbreviation: INH, isonicotinic acid hydrazide. * This material is p a r t of the work t h a t formed the basis of the award of P h . D . Degree b y Madras University in I962 to D. S. R. SARMA.
Biochim. Biophys. Acta, 81 (1964) 3 I I - 3 2 2
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D. S. R. SARMA, S. RAJALAKSHMI, P. S. SARMA
formed is utilized for NAD-biosynthesis via the PREISS AND HANDLER pathway s. The only important biological source for nicotinamide arises from the action of NADglycohydrolase on NAD. The biological significance of these two enzymes, like any other, depends on its universal occurrance, stoichiometry of the reaction and finally on its substrate specificity. For the operation of a cyclic pathway of NAD-synthesis as suggested earlier ~, the two enzymes, nicotinamide amidase and NAD-glycohydrolase should not only occur together but also be very specific towards the respective substrates. The present communication deals with the partial purification of these two enzymes and their substrate specificity. Further, the possible biological significance in the mold Aspergillus niger has been discussed. EXPERIMENTAL
The mold A. niger was maintained by fortnightly subculture on an agar medium 4 and Leuconostoc mesenteroides 9135 was maintained as detailed by JOHNSON9.
Protein determination Protein was precipitated by trichloroacetic acid (lO% final concentration), and centrifuged after an incubation period of 15 min at 4 °. The precipitate was washed twice with lO% trichloroacetic acid. The protein thus obtained was taken in o.I N hot sodium hydroxide and estimated by LOWRY'S method using Folin's reagent 1°.
Purification and properties of nicotinamide amidase Preparation of cell-free extracts : The mold was grown on a 15 % sucrose and inorganic salts medium 4 (pH 3.o) at 3 o°. The four day old mats were washed thoroughly with 0.9% cold saline, pressed free of moisture and frozen at --5 ° for I h. The frozen mats were then cut into small bits and ground with equal amounts of alumina, in a chilled mortar. A lO% homogenate in o.I M sodium phosphate buffer (pH 7.5) was prepared, and centrifuged at 3000 rev./min for IO min to remove alumina and cell debris. The turbid supernatant was used as the enzyme source for further purification. Determination of enzyme activity: The incubation mixture consisted of i.o ml of the enzyme preparation, 800 #g of nicotinamide in water and o.5 ml of o. I M phosphate buffer (pH 7.5) in a final volume of 2 ml. After I h incubation period at 37 °, the reaction was arrested b y keeping the tubes in a boiling water bath for 2 min. The nicotinic acid formed was determined by the microbiological method using L. mesenteroides 9135 as the test organism 9. A unit of activity was defined as #g of nicotinic acid liberated per mg of enzyme preparation per hour. Purification of the enzyme: The enzyme was purified by the conventional methods like ammonium sulphate fractionation, calcium phosphate gel adsorption and sephadex-gel filtration. The enzyme was 57-fold purified and a 59% recovery was achieved. The preparation is free from NAD-giycohydrolase activity. A flow sheet of the various steps of purification has been presented in Scheme I and the results are given in Table I. Biochim. Biophys. Acta, 81 (1964) 311-322
ENZYMES OF N A D METABOLISM IN A .
niger
313
Scheme i Steps involved in the purification of nicotinamide alrddase from A . niger Crude extract (ioo ml) (lO% homogenate in o.I M ~hosphate buffer (pH 7.5))
Ammonium sulphate fractionation (0-50 % saturation)
Supernatant (115 ml)
Precipitate (discarded)
Ammonium sulphate fractionation (50-90%) saturation
+
+ Supernatant (discarded)
Precipitate Dissolved in IOO ml of o.I M phosphate buffer (pH 7-5), dialyzed against o.oi M phosphate buffer (pH 7.5) containing i o - 4 M MgSO4, then centrifuged. The supernatant was treated for 15 rain with 4,6 g of calcium phosphate gel.
+ Precipitate (discarded)
Supernatant Subjected to sephadex G-75 (2o × 2 cm column) gel filtration. The column was equilibrated with o.o 5 M sodium phosphate buffer (pH 7.2.) 5-ml fractions were collected. Material collected in 26-35-ml fraction exhibited the maximum activity.
TABLE I PURIFICATION OF NICOTINAMIDE AMIDASE FROM Aspergillus niger
Crude extract Ammonium sulphate fraction (0-5o%) Ammonium sulphate fraction (50-90%) Dialysis Calcium phosphate gel fraction Sephadex G-75 treatment
Unitslg of wet material
Protein (rag)
3000 IOO 2700 2 IOO 2ooo 178o
60.00 39.oo 6.60 5.oo 1.15 o.63o
Specific activity (unitslrag protein)
50 3 409 42o 1739 2825
Recovery (%)
-3.3 90.0 70.o 66.7 59-3
Purification
-8-fold 8-fold 35-fold 56-5 -f°ld
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D. S. R. SARMA, S. RAJALAKSHMI, P. S SARMA RESULTS
Nicotinamide amidase activity of A. niger was present solely in the supernatant fraction obtained after spinning at ioo ooo × g in a Spinco model L high speed centrifuge. This enzyme hydrolyzed nicotinamide to liberate equimolecular amounts ot nicotinic acid and ammonia. The enzyme exhibited high activity at p H 7.5 and at 4 o°. The enzyme activity was proportional to the substrate concentration, upto 200 #g of nicotinamide. Above 200 Fg and upto 2000 Fg of nicotinamide, there was no further increase in the activity, showing that the enzyme was saturated with the substrate. The Km value of this enzyme for nicotinamide was found to be 6.5" lO -4 M (Fig. I).
1/v 0.03 0.03 0.02 O.02 0.01 0.01~ 0.00!
0
0.;1
Q02
' 0.03
' 0.04
Fig. i. LINEWEAVER--BURK plot for Aspergillus niger nicotinamide amidase. S u b s t r a t e concent r a t i o n expressed as/zg]2 ml. Initial velocity of the reaction expressed as Fg nicotinic acid released in i h. K m = 6.5o 4" IO- I M.
Effect of metal chelating agents The results obtained on the effect of metal chelating agents on the enzyme activity were presented in Table II. From the results it is clear that a,a'-dipyridyl, EDTA, 8-hydroxyquinoline, and sodium diethyl dithiocarbomide inhibited the nicotinamide amidase activity. However, potassium cyanide did not inhibit the enzyme activity. These results pointed to the possible metal ion activation. Among the metal ions tested to reverse the a,a'-dipyridyl inhibition, only Mgz+ reversed the a,a'-dipyridyl inhibition (Table III). In a similar study, the enzyme was incubated with a,a'-dipyridyl for 15 rain and dialysed against o.ooi M Tris buffer (pH 7.5). Marked decrease in activity was observed which was however, restored b y the addition of Mg 2+. The above results suggest the specific dependence of the enzyme reaction on Mg 2÷.
Substrate specificity of nicotinamide amidase Various substances possessing -CO-NH 2 group were tested as probable substrates. It was found that A. niger nicotinamide amidase did not deamidate NADP, NAD, Biochim. Biophys. Acta, 81 (1964) 311-322
ENZYMES OF N A D METABOLISM
I~ A. niger
315
T A B L E II EFFECT
OF METAL CHELATING
AGENTS
ON NICOTINAMIDE
AMIDASE
ACTIVITY
.Enzynre
aaivity (percent of
Additions
control)
None
I oo
a , a ' - D i p y r i d y l (io -4 M) a , a ' - D i p y r i d y l (io -~ M) E D T A (io -4 M) E D T A (lO -2 M) 8 - H y d r o x y q u i n o l i n e (lO -4 M) 8 - H y d r o x y q u i n o l i n e (io-* M) S o d i u m azide (io-* M) S o d i u m azide ( I o ~ M ) S o d i u m d i e t h y l d i f h i o c a r b a m i d e (lO -4 M) S o d i u m d i e t h y l d i t h i o c a r b a m i d e (io -~ M) P o t a s s i u m c y a n i d e (io-* M) P o t a s s i u m c y a n i d e (io -~ M)
46 32 72 58 90 86 89 68 61 32 96 94
NMN, N'-methyl nicotinamide, asparagine, glutamine, benzamide, a-naphthalenamide, indoleacetamide. There is thus a high specificity of the enzyme towards it substrate nicotinamide. In experiments, wherein, NADP, NAD, NMN and N'-methy] nicotinamide were used as substrates, the reaction was followed by measuring the absorbancy of their respective cyanide complexes at 340 m# (see ref. II), when asparagine and glutamine were used, the reaction mixture was analyzed for aspartic acid and glutamic acid by paper chromatographic technique TM. Similarly, when benzamide, and indoleacetamide were used, the same procedure was employed to detect the respective acids13,14. TABLE III REVERSAL
OF INHIBITION PRODUCED BY a,aP=DIPYRIDYL ON NICOTINAMIDE AMIDASE SYSTEM
T h e e n z y m e p r e p a r a t i o n w a s p r e - i n c u b a t e d w i t h a,a'-dipyridyl in a final c o n c e n t r a t i o n of lO -2 M for 15 m i n a n d t h e n dialyzed a g a i n s t o.i M Tris buffer (pH 7-5) a t 2 - 4 ° till t h e c o n t e n t s (outside t h e dialysis tube) g a v e f a i n t colour w i t h FeSO 4. T h e r e t e n t a t e w a s p r e - i n c u b a t e d w i t h v a r i o u s m e t a l ions before t h e a d d i t i o n o f t h e s u b s t r a t e . E~Z~m~
Additions ( zo-t M)
~NIo n e
a,a'-Dipyridyl a,a'-Dipyridyl a,a'-Dipyridyl a,a'=Dipyridyl a,a'=Dipyridyl a,a'-Dipyridyl a,a'-Dipyridyl
activity
(percent of control) I O0
+ + + + + +
Mg *+ M n 2+ Co *+ F e z+ Zn ~+ Fe *+
34 85 38 35 35 Nil Ni]
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D. S. R. SARMA, S. R A J A L A K S H M I , P, S. SARMA
Effect of NA D on nicotinamide amidase activity JOSHI AND HANDLER7 have reported that NAD inhibited nicotinamide amidase activity of yeast. Similar experiments were carried out with purified preparation of the enzyme. However, in the present experiments using A. niger nicotinamide amidase no such inhibition was observed even at a level of 20 mg of NAD per ml of the incubation mixture containing 63 keg of enzyme protein. In these experiments radioactive nicotinamide, with 14C label in the carbonyl group, was used, and the reaction was followed by determining the counts on nicotinic acid spot after separation by ascending paper chromatographic technique using n-but anol saturated with water as the solvent system.
Purification and properties of NA D-glycohydrolase Determination of the enzyme activity: The reaction was followed by the fall in absorbancy of NAD-CN complex at 340 mke in a Beckman Model DU spectrophotometer. The reaction mixture consisted of the following: o.i ml of the enzyme preparation in o.I M sodium phosphate buffer (pH 7.5), 200 keg of NAD in 0.2 ml of distilled water and o.I M phosphate buffer (pH 7.5) in a final volume of i ml. All the reactants were equilibrated at 37 ° for 5 rain before starting the reaction. After an incubation period of 8 min at 37 °, the reaction was arrested by the addition of potassium cyanide solution to give a final concentration of I.O M in a volume of 3 ml. One unit of NAD-glycohydrolase activity was taken as mkemoles of NAD disappeared/h/rag of the enzyme. Purification of NAD-glycohydrolase : Preliminary studies have revealed that the acetone powder preparations could retain the enzyme activity for long periods when kept at low temperatures. Further, acetone treatment destroyed nicotinamide amidase activity of this mold. Hence, acetone powder of this mold was prepared as a first step in the purification procedure and further purification was carried out with this material. Using conventional techniques like ammonium sulfate fractionation and calcium phosphate gel adsorption, the enzyme was purified IO9-fold and 59% of the original activity was recovered. A flow sheet of the various steps of purification is given in Scheme 2 and the results are presented in Table IV. TABLE
IV
PURIFICATION OF NAD-GLYCOHYDROLASIE FROM A . n i g e r
Fraction
Crude extract Acetone powder extract 0-40 % ammonium sulphate saturation 40-8o% ammonium sulphate saturation Dialysis C a l c i u m p h o s p h a t e gel a d s o r p t i o n
units/2oo mg of acetone powder or x g material (wet wt.)
Protein (rag)
318 72o 18o ooo
5o.oo 14.4 o
-56.5
--
3. i
--
52. I 51.8 50.2
148 2o0 15o ooo 695 6oo
i o ooo 166 ooo 164 90o 16o ooo
I. 12 I.iO o.23
R e(%) covery
Specific activity (units/rag protein)
Purification
6374 12 5oo
-2-fold -24-fold 24-fold iog-fold
B i o c h i m . B i o p h y s . A c t a , 81 (1964) 3 1 1 - 3 2 2
ENZYMES OF N A D METABOLISM IN A.
niger
317
Scheme 2 Steps involved in the purification of NAD-gt¥cohydrolase. Crude extract (5 g of material (wet wt.))
Acetone precipitation
+ Acetone powder (i g) Taken in IOO ml of o.i M phosphate buffer (pH 7-5) (Supernatant)
0-40 % a m m o n i u m sulfate saturation
+
¢
S u p e r n a t a n t ( i i o ml)
Precipitate (discarded)
40-80% a m m o n m m sulfate saturation
+
+
S u p e r n a t a n t (discarded)
Precipitate t a k e n in I o o m l of o.I M phosphate buffer (pH 7.5), dialyzed, then centrifuged. The s u p e r n a t a n t was treated with 4.6 g of calcium p h o s p h a t e gel.
+
+
S u p e r n a t a n t (used as the enzyme source)
Precipitate (discarded)
Stoichiometry of the reaction: In the present experiments, the point of enzymic attack of NAD was based on the following observations. (a) Fall in the absorbancy of NAD-CN complex at 325 m/z or 340 m/~. (b) Liberation ofnicotinamide as one of the reaction products. (c) Inability of the enzyme preparation to attack NMN. The fall in absorbancy at 325 m# indicates that the split m a y be either at the amide group or at the nicotinamide riboside bridge in NAD. The fact that equimolecular concentrations of nicotinamide could be detected as one of the reaction products, clearly indicates that the cleavage was at the nicotinamide-riboside link. This observation was further confirmed by the identification of nicotinic acid as one of the reaction products, when the reaction mixture was treated with the purified A. niger nicotinamide amidase. The fact that the enzyme preparation did not attack NMN rules out the possibility that the cleavage of nicotinamide riboside link in NAD occurred after the breakdown of the pyrophosphate bridge of NAD. All the above observations reveal that the enzyme split was at the nicotinamide-riboside linkin NAD. Having established the point of attack, the stoichiometry of the reaction was studied. The results presented Biochim. Biophys. Acta, 81 (I964) 3 i i - 3 2 2
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D. S. R. SARMA, S. R A J A L A K S H M I , P. S. S A R M A
in Table V show t h a t for every one molecule of NAD disappeared one molecule ot nicotinamide was liberated. N A D disappearance was followed b y measuring the absorbancy of the N A D - C N complex at 34o m/~ and the nicotinamide was determined in the protein-free filtrates by the cyanogen bromide method 15. Protein-free filtrates were obtained b y treating the enzyme reaction mixtures with equal parts of barium hydroxide and zinc sulfatO ~. TABLE V STOICHIOMETRY
Experimental system
OF T H E E N Z Y M E R E A C T I O N
N A D consumed (#moles)
Blank Test
Nil 2.658
Nicotinamide released (/*moles)
Mole N A D consumed Mole nieotinamide released
Nil 2.620
-i.Ol 4
Stability of the enzyme: The final purified preparation could be kept in the deepfreeze for weeks without loss of activity. Similarly, negligible loss of activity was observed when the acetone powders were kept for 18-24 months in the deep-freeze at - - 20 °. Since per unit volume of the basal medium A. niger gives a mat which weighs ten times greater than t h a t of Neurospora, it might be t h a t for large scale preparation of NAD-glycohydrolase A. niger will serve a better source than Neurospora mold. Properties of NAD-glycohydrolase: The enzyme activity was found to be in the IOO ooo × g supernatant, and exhibited highest activity over a p H range of 7.5-9.0 and at 4 o°. The activity was linear upto io rain. The enzyme activity was proportional to the substrate concentration upto a concentration of 80/,moles of NAD. The Km value of this enzyme for N A D was found to be 7.693" lO -6 M (Fig. 2).
0.05
0.0~ OD3
0.0;
0.01
o
o.o~
0.02 0.03
o.~
o~
1~Is] Fig. 2. LINEWEAVER--BURKplot of NAD-glycohydrolase. Substrate concentration expressed as m/~moles/ml. Initial velocity of the reaction expressed as m#moles of NAD disappeared in 8 rain. K
m
=
7.693" IO -5
M.
A. niger NAD-glycohydrolase activity was not inhibited b y any of the metal chelating agents tested such as E D T A , a,atdipyridyl, o-phenanthroline, sodium azide and fluoride (all at 10 -2 M final concentration), thus suggesting t h a t this enzyme did Biochira. Biophys. Acta, 81 (1964) 311-322
ENZYMES OF NAD
METABOLISM IN A.
niger
319
not require any metal ion for activation. On the other hand except Mg2+, other meta] ions like Mn 2+, Cos+, Zn ~+, Fe z+, Fe 3+ and Hg 2+ (all at lO -2 M final concentration) inhibited the enzyme activity. In these experiments, the incubation was carried out in o.I M Tris buffer (pH 7.5) and the reaction was followed by the fluorimetric method and not by cyanide dadition, since cyanide addition resulted in intense colouration with Mn 2+, Fe ~÷ and Fe 8+, which exhibited high absorption at 340 InF. Effect of pyridine compounds on NAD-glycohydrolase: It is known that animal NAD-glycohydrolases are sensitive to either nicotinamide or INH. Neurospora NADglycohydrolase was not inhibited appreciably by nicotinamide. However, in the presence of ergothionine, nicotinamide could inhibit the enzyme activity. In the present experiment, the effect of nicotinic acid, its amide and I N H on NAD-glycohydrolase was studied. The results presented in Table VI clearly indicate that at the levels tried, none of these compounds could inhibit the enzyme activity. TABLE VI EFFECT OF ADENINE, ADENOSINE,
NICOTINICA C I D
ADP,
AND INH
RIBOSE, RIBOSE 5-PHOSPHATE, NICOTINAMIDE, ON
NAD-GLYCOHYDROLASE
The enzyme was preincubated with the above compounds for IO rnin at 37° before adding the substrate. Various concentrations of these substances were used. The results obtained with the highest concentration (io -* M) are presented here. A dditiens M)
(at z o -=
None Adenine Adenosine ADP Ribose
Rib-5-P
Nicotinamide Nicotinic acid INH
gn..yme activity (mi*moles/ h)
935 985 960 985 97° 885 91o 90o 900
Attempts were also made to study the sites of attachment of NAD to the enzyme surface. Accordingly the effect of various components of NAD such as adenine, adenosine, ADP, ATP, ribose, Rib-5-P, nicotinamide on NAD-glycohydrolase activity was studied. It is evident from the results presented in Table VI that none of the compounds tested either independently or in combination could inhibit the enzyme reaction. However, the possibility of higher concentrations of these compounds inhibiting the enzyme reaction cannot be ruled out. Substrate specificity of A. niger NAD-glycohydrolase: NAD-glycohydrolases in general, exhibited substrate specificity. Most of the NAD-glycohydrolases studied attacked mainly NAD and NADP. Using sheep-brain NAD-glycohydrolase it was shown that substrates such as nicotinamide ribofuranoside, nicotinamide glycoside and other nicotinamide derivatives were not attacked 1~. NAD-glycohydrolase obtained from Neurospora could cleave only NAD and NADP. In these experiments it was observed, that while the final purified preparation could act only on NAD and NADP, it failed to attack NMN, N-methylnicotinamide, and NADH (Table vii). Further, the enzyme did not act on tryptamine-NAD complex as efficiently as on NAD molecule.
Biochim. Biophys. Acta, 81 (i964) 311-322
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D.S.R.
SARMA, S. RAJALAKSHMI, P. S. SARMA TABLE VII
SUBSTRATE SPECIFICITY OF 2J. niger NAD-GLYCOHYDROLASE Substrate
Enzyme activity (mlzmoles/h)
NAD NADP NMN N ' - m e t h y l nicotinamide NADH
753.36 724.26 20.40 24.6o 6.o2
The interaction ofindoles and NAD was studied in some detail by ALIVISATOSet al. 18-2°. These authors observed that the pyrrolic nitrogen of the indoles acted as a nucleophilic agent, attacking an electrophilic centre in NAD, probably C-4 ofnicotinamide. Further, they have reported that such a complex inhibited NAD-glycohydrolase activity. In the present experiments tryptamine was used. Tryptamine was selected, because it was relatively more soluble than tryptophane and more stable than serotonin. Equimolar amounts of NAD and tryptamine hydrochloride were incubated at room temperature (25 °) for i h and then the enzyme was added. The results presented in Table VIII reveal that tryptamine-NAD complex was not acted upon by NADglycohydrolase. In this respect A. niger NAD-glycohydrolase has a similar specificity as that of animal NAD-glycohydrolase. TABLE VIII EFFECT
OF
TRYPTAMINE oN NAD-GLYCOHYDROLASE
2/*moles of N A D and t r y p t a m i n e at the concentrations mentioned above were incubated for I h at 25 ° and t h e n o.i ml of the enzyme p r e p a r a t i o n was added, NAD-glycohydrolase was assayed b y measuring the a b s o r p t i o n of the cyanide complex at 34 ° m/*. Addition
None T r y p t a m i n e (2/*moles) T r y p t a m i n e (4/*moles)
Enzyme activity (ml*moleslh) 785 664 372
Inhibition (%)
-15 53
From the above results it is evident that the following characteristic groupings in NAD molecule are essential for NAD-glycohydrolase to act upon: (a) complete molecule of NAD, (b) quarternary nitrogen of nicotinamide molecule, (c) benzenoid structure of nicotinamide molecule, (d) free C-4 of nicotinamide, (e) free 6-amino group of adenine 16.
Biological role of NAD-glycohydrolase and nicotinamide amidase in the regulation of cellular NAD-concentration The object of purifying these two enzymes has been to study the substrate specificity and understand the biological significance of these two enzymes. The results presented in this communication revealed that both the enzymes, nicotinamide Biochim. Biophys. Aeta, 81 (1964) 311-322
ENZYMES OF N A D METABOLISM IN A .
niger
321
amidase and NAD-glycohydrolase of A. niger are specific towards their respective substrates. The stoichiometry of the enzyme reactions was also established. Further methylation of nicotinamide and amidation of nicotinic acid could not be demonstrated in this mold 21. In view of the presence of nicotinic acid pathway of NAD synthesis 8, it was suggested that nicotinamide amidase plays an important role in deamidating nicotinamide to nicotinic acid, which can be reutilized for NAD synthesis. A potent biological source for nicotinamide would thus be obligatory for the operation of such a p a t h w a y in the cell. In the absence of amidation of nicotinic acid to nicotinamide 21, the only biological source in this organism appears to be b y the action of NADglycohydrolase on NAD. Further this mold exhibited the presence of both nicotinamide amidase and NAD-glycohydrolase activities. The presence of these two highly specific enzymes occurring together and the absence of other metabolic pathways for nicotinamide in this organism support the suggested cyclic pathway of NAD synthesis 8, according to which the liberated nicotinamide by the action of NAD-glycohydrolase on NAD gets deamidated to nicotinic acid which could be reutilized for NAD synthesis. In yeast JOSHI AND HANDLER7 have reported that NAD at and above certain concentration inhibits nicotinamide amidase. Thus when the concentration of cellular NAD increased it inhibited the nicotinamide amidase and thereby arrested the NADsynthesis from nicotinamide via nicotinic acid. Since the microbial NAD-glycohydrolases such as NAD-glycohydrolase of yeast is not inhibited by nicotinamide, the NADglycohydrolase will act upon the NAD and liberate nicotinamide, with a corresponding decrease in the level of cellular NAD. When the level of cellular NAD goes below the critical concentration, at which it can no longer inhibit nicotinamide amidase, then the liberated nicotinamide is reutilized for NAD synthesis via nicotinic acid, untilit reached a concentration sufficient for inhibiting the nicotinamide amidase. However, using purified preparation of nicotinamide amidase from A. niger it was observed that NAD at a level of 20 mg did not inhibit this enzyme activity to any great extent. Since nicotinamide at higher concentration was toxic to the growth of the mold 21, such a feedback mechanism as suggested by JosHI AND HANDLER~ in yeast m a y not be favourable to the mold A. niger. Probably NAD might exert a feedback control on nicotinic acid mononucleotide pyrophosphorylase (nicotinic acid mononucleotide: pyrophosphate phosphoribosyltransferase) rather than on nicotinamide amidase, an enzyme in the salvage p a t h w a y of NAD synthesis. ACKNOWLEDGEMENTS
Financial aid to D. S. R. SARMAand S. RAJALAKSHMIin the form of Senior Research Fellowships by the Council of Scientific and Industrial Research, New Delhi, is gratefully acknowledged. REFERENCES 1 D. E. HUGHES AND D. H. WILLIAMSON, Biochem. J., 55 (I953) 85 I. * Y. OEA, J. Biochem. Tokyo, 41 (1954) 89. 8 y . S. HALPERN AND H. GROSSOWlCZ, Biochem. J., 65 (1957) 716. * K. V. RAJAGOPALAN, D. S. R. SARMA, T. K. SUNDARAM AND P. S. SARMA, Enzymologia, 21 (1959) 277. 5 K. V. RAJAGOPALAN, T. K. SUNDARAM AND P. S. SARMA, Nature, 184 (1959) 461. 6 D. S. R. SARMA, S. RAJALAKSHMI AND P. S. SARMA, Biochem. Biophys. Res. Commun., 6 (1961) 389 •
Biochim. Biophys. Acta, 81 (I964) 311-322
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D. S. R. SARMA, S. RAJALAKSHMI, P. S. SARMA
7 j . C. JOSHI AND P. HANDLER, J. Biol. Chem., 237 (1962) 929. s j . PREISS AND P. HANDLER, J. Biol. Chem., 233 (1958) 493. 9 ]3. C. JOHNSON, J. Biol. Chem., 159 (1945) 227. 10 C. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 11 S. P. COLOWlCK, N. O. KAPLAN AND N. M. CIOTTI, J. Biol. Chem., 191 (1951) 447. 12 p. DEEKER AND W. RIFFART, Chem. Ztg., 74 (195 °) 261. la M. E. FEWSTER AND D. A. HALL, Nature, 168 (1951) 78. 14 A. J. VLITOS AND W. MEUDT, Contrib. Boyce Thomson Inst., 17 (1953) 197. 15 H. MclLwAIN AND R. RODNIGHT, Biochem. J., 44 (1949) 47 °. 16 lX]'. O. KAPLAN, S. P. COLOWICK AND A. NASON, J. Biol. Chem., 191 (1951) 473. 1~ H. MclLwAIN AND R. RODNIGHT, Biochem. J., 45 (1949) 337xs S. G. A. ALIVlSATOS, Nature, 183 (1959) lO34. 19 S. G. A. ALIVlSATOS, G. A. MOURKIDES AND A. JIBRIL, Nature, 196 (196o) 718. 2o S. G. A. ALIVISATOS, F. UNGAR, A. JIBRIL AND G. A. MOURKIDES, Biochim. Biophys. Acta 51 (1961) 361. 21 D. S. R. SARMA, S. I~_AJALAKSHMIAND P. S. SARMA, Enzymologia, 24 (1962) 148.
Biochim. Biophys. Acta, 81 (1964) 311-32
311
BBA 1233O S T U D I E S ON T H E E N Z Y M E S I N V O L V E D I N N I C O T I N A M I D E A D E N I N E D I N U C L E O T I D E M E T A B O L I S M IN
A S P E R G I L L US NIGER* D. S. R. SARMA, S. R A J A L A K S H M I AND P. S. SARMA
Department of Biochemistry, Indian Institute of Science, Bangalore (India) (Received J u n e 4th, 1963)
SUMMARY
The enzyme nicotinamide amidase (n~.cotinamide amidohydrolase) was purified 57-fold from Aspergillus niger. The purified preparation was specific towards its substrate nicotinamide and did not deamidate NADP, NAD, NMN, N'-methyl nicotinamide, asparagine, glutamine, benzamide, a-naphthaleneamide and indoleacetamide. The optimum pH was found to be 7.5. Temperature optimum was 4 o°. It had a Km value of 6.504. lO -4 M towards nicotinamide. The enzyme exhibited Mg~+ ion requirement for its optimum activity. NAD-glycohydrolase (EC 3.2.2.5) was purified Iog-fold from the mold A. niger. The enzyme preparation was active only towards NAD and NADP and did not attack NMN, N'-methylnicotinamide and NADH. The Km value for NAD was found to be 7.693. lO -6 M. The enzyme did not require any metal ion for its activity. It is suggested that A. niger will serve a better source for a large scale preparation of NAD-glycohydrolase than the Neurospora mold. The biological role of both NAD-glycohydrolase and nicotinamide amidase in the regulation of cellular NAD level has been discussed. It is, further, observed that NAD did not exert its feedback control on nicotinamide amidase at least in A. niger. INTRODUCTION
Nicotinamide amidase (nicotinamide amidohydrolase), an hydrolysing enzyme involved in NAD metabolism, was first demonstrated in lactic acid bacteria by HUGHES AND WILLIAMSON 1. Later the enzyme was found to be present in microorganisms 9-4 as well as in higher vertebrate tissues 5. However, the biological significance of this hydrolysing enzyme is yet to be understood. Only in recent years the role of nicotinamide amidase and NAD-glycohydrolase (EC 3.2.2.5) in the maintenance of cellular NAD level has been studiede, 7. According to these investigations the enzyme "nicotinamide amidase" deamidates nicotinamide to nicotinic acid and the nicotinic acid Abbreviation: INH, isonicotinic acid hydrazide. * This material is p a r t of the work t h a t formed the basis of the award of P h . D . Degree b y Madras University in I962 to D. S. R. SARMA.
Biochim. Biophys. Acta, 81 (1964) 3 I I - 3 2 2
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D. S. R. SARMA, S. RAJALAKSHMI, P. S. SARMA
formed is utilized for NAD-biosynthesis via the PREISS AND HANDLER pathway s. The only important biological source for nicotinamide arises from the action of NADglycohydrolase on NAD. The biological significance of these two enzymes, like any other, depends on its universal occurrance, stoichiometry of the reaction and finally on its substrate specificity. For the operation of a cyclic pathway of NAD-synthesis as suggested earlier ~, the two enzymes, nicotinamide amidase and NAD-glycohydrolase should not only occur together but also be very specific towards the respective substrates. The present communication deals with the partial purification of these two enzymes and their substrate specificity. Further, the possible biological significance in the mold Aspergillus niger has been discussed. EXPERIMENTAL
The mold A. niger was maintained by fortnightly subculture on an agar medium 4 and Leuconostoc mesenteroides 9135 was maintained as detailed by JOHNSON9.
Protein determination Protein was precipitated by trichloroacetic acid (lO% final concentration), and centrifuged after an incubation period of 15 min at 4 °. The precipitate was washed twice with lO% trichloroacetic acid. The protein thus obtained was taken in o.I N hot sodium hydroxide and estimated by LOWRY'S method using Folin's reagent 1°.
Purification and properties of nicotinamide amidase Preparation of cell-free extracts : The mold was grown on a 15 % sucrose and inorganic salts medium 4 (pH 3.o) at 3 o°. The four day old mats were washed thoroughly with 0.9% cold saline, pressed free of moisture and frozen at --5 ° for I h. The frozen mats were then cut into small bits and ground with equal amounts of alumina, in a chilled mortar. A lO% homogenate in o.I M sodium phosphate buffer (pH 7.5) was prepared, and centrifuged at 3000 rev./min for IO min to remove alumina and cell debris. The turbid supernatant was used as the enzyme source for further purification. Determination of enzyme activity: The incubation mixture consisted of i.o ml of the enzyme preparation, 800 #g of nicotinamide in water and o.5 ml of o. I M phosphate buffer (pH 7.5) in a final volume of 2 ml. After I h incubation period at 37 °, the reaction was arrested b y keeping the tubes in a boiling water bath for 2 min. The nicotinic acid formed was determined by the microbiological method using L. mesenteroides 9135 as the test organism 9. A unit of activity was defined as #g of nicotinic acid liberated per mg of enzyme preparation per hour. Purification of the enzyme: The enzyme was purified by the conventional methods like ammonium sulphate fractionation, calcium phosphate gel adsorption and sephadex-gel filtration. The enzyme was 57-fold purified and a 59% recovery was achieved. The preparation is free from NAD-giycohydrolase activity. A flow sheet of the various steps of purification has been presented in Scheme I and the results are given in Table I. Biochim. Biophys. Acta, 81 (1964) 311-322
ENZYMES OF N A D METABOLISM IN A .
niger
313
Scheme i Steps involved in the purification of nicotinamide alrddase from A . niger Crude extract (ioo ml) (lO% homogenate in o.I M ~hosphate buffer (pH 7.5))
Ammonium sulphate fractionation (0-50 % saturation)
Supernatant (115 ml)
Precipitate (discarded)
Ammonium sulphate fractionation (50-90%) saturation
+
+ Supernatant (discarded)
Precipitate Dissolved in IOO ml of o.I M phosphate buffer (pH 7-5), dialyzed against o.oi M phosphate buffer (pH 7.5) containing i o - 4 M MgSO4, then centrifuged. The supernatant was treated for 15 rain with 4,6 g of calcium phosphate gel.
+ Precipitate (discarded)
Supernatant Subjected to sephadex G-75 (2o × 2 cm column) gel filtration. The column was equilibrated with o.o 5 M sodium phosphate buffer (pH 7.2.) 5-ml fractions were collected. Material collected in 26-35-ml fraction exhibited the maximum activity.
TABLE I PURIFICATION OF NICOTINAMIDE AMIDASE FROM Aspergillus niger
Crude extract Ammonium sulphate fraction (0-5o%) Ammonium sulphate fraction (50-90%) Dialysis Calcium phosphate gel fraction Sephadex G-75 treatment
Unitslg of wet material
Protein (rag)
3000 IOO 2700 2 IOO 2ooo 178o
60.00 39.oo 6.60 5.oo 1.15 o.63o
Specific activity (unitslrag protein)
50 3 409 42o 1739 2825
Recovery (%)
-3.3 90.0 70.o 66.7 59-3
Purification
-8-fold 8-fold 35-fold 56-5 -f°ld
Biochim. Biophys. Acta, 81 (1964) 3II-32~
314
D. S. R. SARMA, S. RAJALAKSHMI, P. S SARMA RESULTS
Nicotinamide amidase activity of A. niger was present solely in the supernatant fraction obtained after spinning at ioo ooo × g in a Spinco model L high speed centrifuge. This enzyme hydrolyzed nicotinamide to liberate equimolecular amounts ot nicotinic acid and ammonia. The enzyme exhibited high activity at p H 7.5 and at 4 o°. The enzyme activity was proportional to the substrate concentration, upto 200 #g of nicotinamide. Above 200 Fg and upto 2000 Fg of nicotinamide, there was no further increase in the activity, showing that the enzyme was saturated with the substrate. The Km value of this enzyme for nicotinamide was found to be 6.5" lO -4 M (Fig. I).
1/v 0.03 0.03 0.02 O.02 0.01 0.01~ 0.00!
0
0.;1
Q02
' 0.03
' 0.04
Fig. i. LINEWEAVER--BURK plot for Aspergillus niger nicotinamide amidase. S u b s t r a t e concent r a t i o n expressed as/zg]2 ml. Initial velocity of the reaction expressed as Fg nicotinic acid released in i h. K m = 6.5o 4" IO- I M.
Effect of metal chelating agents The results obtained on the effect of metal chelating agents on the enzyme activity were presented in Table II. From the results it is clear that a,a'-dipyridyl, EDTA, 8-hydroxyquinoline, and sodium diethyl dithiocarbomide inhibited the nicotinamide amidase activity. However, potassium cyanide did not inhibit the enzyme activity. These results pointed to the possible metal ion activation. Among the metal ions tested to reverse the a,a'-dipyridyl inhibition, only Mgz+ reversed the a,a'-dipyridyl inhibition (Table III). In a similar study, the enzyme was incubated with a,a'-dipyridyl for 15 rain and dialysed against o.ooi M Tris buffer (pH 7.5). Marked decrease in activity was observed which was however, restored b y the addition of Mg 2+. The above results suggest the specific dependence of the enzyme reaction on Mg 2÷.
Substrate specificity of nicotinamide amidase Various substances possessing -CO-NH 2 group were tested as probable substrates. It was found that A. niger nicotinamide amidase did not deamidate NADP, NAD, Biochim. Biophys. Acta, 81 (1964) 311-322
ENZYMES OF N A D METABOLISM
I~ A. niger
315
T A B L E II EFFECT
OF METAL CHELATING
AGENTS
ON NICOTINAMIDE
AMIDASE
ACTIVITY
.Enzynre
aaivity (percent of
Additions
control)
None
I oo
a , a ' - D i p y r i d y l (io -4 M) a , a ' - D i p y r i d y l (io -~ M) E D T A (io -4 M) E D T A (lO -2 M) 8 - H y d r o x y q u i n o l i n e (lO -4 M) 8 - H y d r o x y q u i n o l i n e (io-* M) S o d i u m azide (io-* M) S o d i u m azide ( I o ~ M ) S o d i u m d i e t h y l d i f h i o c a r b a m i d e (lO -4 M) S o d i u m d i e t h y l d i t h i o c a r b a m i d e (io -~ M) P o t a s s i u m c y a n i d e (io-* M) P o t a s s i u m c y a n i d e (io -~ M)
46 32 72 58 90 86 89 68 61 32 96 94
NMN, N'-methyl nicotinamide, asparagine, glutamine, benzamide, a-naphthalenamide, indoleacetamide. There is thus a high specificity of the enzyme towards it substrate nicotinamide. In experiments, wherein, NADP, NAD, NMN and N'-methy] nicotinamide were used as substrates, the reaction was followed by measuring the absorbancy of their respective cyanide complexes at 340 m# (see ref. II), when asparagine and glutamine were used, the reaction mixture was analyzed for aspartic acid and glutamic acid by paper chromatographic technique TM. Similarly, when benzamide, and indoleacetamide were used, the same procedure was employed to detect the respective acids13,14. TABLE III REVERSAL
OF INHIBITION PRODUCED BY a,aP=DIPYRIDYL ON NICOTINAMIDE AMIDASE SYSTEM
T h e e n z y m e p r e p a r a t i o n w a s p r e - i n c u b a t e d w i t h a,a'-dipyridyl in a final c o n c e n t r a t i o n of lO -2 M for 15 m i n a n d t h e n dialyzed a g a i n s t o.i M Tris buffer (pH 7-5) a t 2 - 4 ° till t h e c o n t e n t s (outside t h e dialysis tube) g a v e f a i n t colour w i t h FeSO 4. T h e r e t e n t a t e w a s p r e - i n c u b a t e d w i t h v a r i o u s m e t a l ions before t h e a d d i t i o n o f t h e s u b s t r a t e . E~Z~m~
Additions ( zo-t M)
~NIo n e
a,a'-Dipyridyl a,a'-Dipyridyl a,a'-Dipyridyl a,a'=Dipyridyl a,a'=Dipyridyl a,a'-Dipyridyl a,a'-Dipyridyl
activity
(percent of control) I O0
+ + + + + +
Mg *+ M n 2+ Co *+ F e z+ Zn ~+ Fe *+
34 85 38 35 35 Nil Ni]
Biochim. Biophys. Acfa, 8i (i964) 3 i i - 3 2 2
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D. S. R. SARMA, S. R A J A L A K S H M I , P, S. SARMA
Effect of NA D on nicotinamide amidase activity JOSHI AND HANDLER7 have reported that NAD inhibited nicotinamide amidase activity of yeast. Similar experiments were carried out with purified preparation of the enzyme. However, in the present experiments using A. niger nicotinamide amidase no such inhibition was observed even at a level of 20 mg of NAD per ml of the incubation mixture containing 63 keg of enzyme protein. In these experiments radioactive nicotinamide, with 14C label in the carbonyl group, was used, and the reaction was followed by determining the counts on nicotinic acid spot after separation by ascending paper chromatographic technique using n-but anol saturated with water as the solvent system.
Purification and properties of NA D-glycohydrolase Determination of the enzyme activity: The reaction was followed by the fall in absorbancy of NAD-CN complex at 340 mke in a Beckman Model DU spectrophotometer. The reaction mixture consisted of the following: o.i ml of the enzyme preparation in o.I M sodium phosphate buffer (pH 7.5), 200 keg of NAD in 0.2 ml of distilled water and o.I M phosphate buffer (pH 7.5) in a final volume of i ml. All the reactants were equilibrated at 37 ° for 5 rain before starting the reaction. After an incubation period of 8 min at 37 °, the reaction was arrested by the addition of potassium cyanide solution to give a final concentration of I.O M in a volume of 3 ml. One unit of NAD-glycohydrolase activity was taken as mkemoles of NAD disappeared/h/rag of the enzyme. Purification of NAD-glycohydrolase : Preliminary studies have revealed that the acetone powder preparations could retain the enzyme activity for long periods when kept at low temperatures. Further, acetone treatment destroyed nicotinamide amidase activity of this mold. Hence, acetone powder of this mold was prepared as a first step in the purification procedure and further purification was carried out with this material. Using conventional techniques like ammonium sulfate fractionation and calcium phosphate gel adsorption, the enzyme was purified IO9-fold and 59% of the original activity was recovered. A flow sheet of the various steps of purification is given in Scheme 2 and the results are presented in Table IV. TABLE
IV
PURIFICATION OF NAD-GLYCOHYDROLASIE FROM A . n i g e r
Fraction
Crude extract Acetone powder extract 0-40 % ammonium sulphate saturation 40-8o% ammonium sulphate saturation Dialysis C a l c i u m p h o s p h a t e gel a d s o r p t i o n
units/2oo mg of acetone powder or x g material (wet wt.)
Protein (rag)
318 72o 18o ooo
5o.oo 14.4 o
-56.5
--
3. i
--
52. I 51.8 50.2
148 2o0 15o ooo 695 6oo
i o ooo 166 ooo 164 90o 16o ooo
I. 12 I.iO o.23
R e(%) covery
Specific activity (units/rag protein)
Purification
6374 12 5oo
-2-fold -24-fold 24-fold iog-fold
B i o c h i m . B i o p h y s . A c t a , 81 (1964) 3 1 1 - 3 2 2
ENZYMES OF N A D METABOLISM IN A.
niger
317
Scheme 2 Steps involved in the purification of NAD-gt¥cohydrolase. Crude extract (5 g of material (wet wt.))
Acetone precipitation
+ Acetone powder (i g) Taken in IOO ml of o.i M phosphate buffer (pH 7-5) (Supernatant)
0-40 % a m m o n i u m sulfate saturation
+
¢
S u p e r n a t a n t ( i i o ml)
Precipitate (discarded)
40-80% a m m o n m m sulfate saturation
+
+
S u p e r n a t a n t (discarded)
Precipitate t a k e n in I o o m l of o.I M phosphate buffer (pH 7.5), dialyzed, then centrifuged. The s u p e r n a t a n t was treated with 4.6 g of calcium p h o s p h a t e gel.
+
+
S u p e r n a t a n t (used as the enzyme source)
Precipitate (discarded)
Stoichiometry of the reaction: In the present experiments, the point of enzymic attack of NAD was based on the following observations. (a) Fall in the absorbancy of NAD-CN complex at 325 m/z or 340 m/~. (b) Liberation ofnicotinamide as one of the reaction products. (c) Inability of the enzyme preparation to attack NMN. The fall in absorbancy at 325 m# indicates that the split m a y be either at the amide group or at the nicotinamide riboside bridge in NAD. The fact that equimolecular concentrations of nicotinamide could be detected as one of the reaction products, clearly indicates that the cleavage was at the nicotinamide-riboside link. This observation was further confirmed by the identification of nicotinic acid as one of the reaction products, when the reaction mixture was treated with the purified A. niger nicotinamide amidase. The fact that the enzyme preparation did not attack NMN rules out the possibility that the cleavage of nicotinamide riboside link in NAD occurred after the breakdown of the pyrophosphate bridge of NAD. All the above observations reveal that the enzyme split was at the nicotinamide-riboside linkin NAD. Having established the point of attack, the stoichiometry of the reaction was studied. The results presented Biochim. Biophys. Acta, 81 (I964) 3 i i - 3 2 2
318
D. S. R. SARMA, S. R A J A L A K S H M I , P. S. S A R M A
in Table V show t h a t for every one molecule of NAD disappeared one molecule ot nicotinamide was liberated. N A D disappearance was followed b y measuring the absorbancy of the N A D - C N complex at 34o m/~ and the nicotinamide was determined in the protein-free filtrates by the cyanogen bromide method 15. Protein-free filtrates were obtained b y treating the enzyme reaction mixtures with equal parts of barium hydroxide and zinc sulfatO ~. TABLE V STOICHIOMETRY
Experimental system
OF T H E E N Z Y M E R E A C T I O N
N A D consumed (#moles)
Blank Test
Nil 2.658
Nicotinamide released (/*moles)
Mole N A D consumed Mole nieotinamide released
Nil 2.620
-i.Ol 4
Stability of the enzyme: The final purified preparation could be kept in the deepfreeze for weeks without loss of activity. Similarly, negligible loss of activity was observed when the acetone powders were kept for 18-24 months in the deep-freeze at - - 20 °. Since per unit volume of the basal medium A. niger gives a mat which weighs ten times greater than t h a t of Neurospora, it might be t h a t for large scale preparation of NAD-glycohydrolase A. niger will serve a better source than Neurospora mold. Properties of NAD-glycohydrolase: The enzyme activity was found to be in the IOO ooo × g supernatant, and exhibited highest activity over a p H range of 7.5-9.0 and at 4 o°. The activity was linear upto io rain. The enzyme activity was proportional to the substrate concentration upto a concentration of 80/,moles of NAD. The Km value of this enzyme for N A D was found to be 7.693" lO -6 M (Fig. 2).
0.05
0.0~ OD3
0.0;
0.01
o
o.o~
0.02 0.03
o.~
o~
1~Is] Fig. 2. LINEWEAVER--BURKplot of NAD-glycohydrolase. Substrate concentration expressed as m/~moles/ml. Initial velocity of the reaction expressed as m#moles of NAD disappeared in 8 rain. K
m
=
7.693" IO -5
M.
A. niger NAD-glycohydrolase activity was not inhibited b y any of the metal chelating agents tested such as E D T A , a,atdipyridyl, o-phenanthroline, sodium azide and fluoride (all at 10 -2 M final concentration), thus suggesting t h a t this enzyme did Biochira. Biophys. Acta, 81 (1964) 311-322
ENZYMES OF NAD
METABOLISM IN A.
niger
319
not require any metal ion for activation. On the other hand except Mg2+, other meta] ions like Mn 2+, Cos+, Zn ~+, Fe z+, Fe 3+ and Hg 2+ (all at lO -2 M final concentration) inhibited the enzyme activity. In these experiments, the incubation was carried out in o.I M Tris buffer (pH 7.5) and the reaction was followed by the fluorimetric method and not by cyanide dadition, since cyanide addition resulted in intense colouration with Mn 2+, Fe ~÷ and Fe 8+, which exhibited high absorption at 340 InF. Effect of pyridine compounds on NAD-glycohydrolase: It is known that animal NAD-glycohydrolases are sensitive to either nicotinamide or INH. Neurospora NADglycohydrolase was not inhibited appreciably by nicotinamide. However, in the presence of ergothionine, nicotinamide could inhibit the enzyme activity. In the present experiment, the effect of nicotinic acid, its amide and I N H on NAD-glycohydrolase was studied. The results presented in Table VI clearly indicate that at the levels tried, none of these compounds could inhibit the enzyme activity. TABLE VI EFFECT OF ADENINE, ADENOSINE,
NICOTINICA C I D
ADP,
AND INH
RIBOSE, RIBOSE 5-PHOSPHATE, NICOTINAMIDE, ON
NAD-GLYCOHYDROLASE
The enzyme was preincubated with the above compounds for IO rnin at 37° before adding the substrate. Various concentrations of these substances were used. The results obtained with the highest concentration (io -* M) are presented here. A dditiens M)
(at z o -=
None Adenine Adenosine ADP Ribose
Rib-5-P
Nicotinamide Nicotinic acid INH
gn..yme activity (mi*moles/ h)
935 985 960 985 97° 885 91o 90o 900
Attempts were also made to study the sites of attachment of NAD to the enzyme surface. Accordingly the effect of various components of NAD such as adenine, adenosine, ADP, ATP, ribose, Rib-5-P, nicotinamide on NAD-glycohydrolase activity was studied. It is evident from the results presented in Table VI that none of the compounds tested either independently or in combination could inhibit the enzyme reaction. However, the possibility of higher concentrations of these compounds inhibiting the enzyme reaction cannot be ruled out. Substrate specificity of A. niger NAD-glycohydrolase: NAD-glycohydrolases in general, exhibited substrate specificity. Most of the NAD-glycohydrolases studied attacked mainly NAD and NADP. Using sheep-brain NAD-glycohydrolase it was shown that substrates such as nicotinamide ribofuranoside, nicotinamide glycoside and other nicotinamide derivatives were not attacked 1~. NAD-glycohydrolase obtained from Neurospora could cleave only NAD and NADP. In these experiments it was observed, that while the final purified preparation could act only on NAD and NADP, it failed to attack NMN, N-methylnicotinamide, and NADH (Table vii). Further, the enzyme did not act on tryptamine-NAD complex as efficiently as on NAD molecule.
Biochim. Biophys. Acta, 81 (i964) 311-322
320
D.S.R.
SARMA, S. RAJALAKSHMI, P. S. SARMA TABLE VII
SUBSTRATE SPECIFICITY OF 2J. niger NAD-GLYCOHYDROLASE Substrate
Enzyme activity (mlzmoles/h)
NAD NADP NMN N ' - m e t h y l nicotinamide NADH
753.36 724.26 20.40 24.6o 6.o2
The interaction ofindoles and NAD was studied in some detail by ALIVISATOSet al. 18-2°. These authors observed that the pyrrolic nitrogen of the indoles acted as a nucleophilic agent, attacking an electrophilic centre in NAD, probably C-4 ofnicotinamide. Further, they have reported that such a complex inhibited NAD-glycohydrolase activity. In the present experiments tryptamine was used. Tryptamine was selected, because it was relatively more soluble than tryptophane and more stable than serotonin. Equimolar amounts of NAD and tryptamine hydrochloride were incubated at room temperature (25 °) for i h and then the enzyme was added. The results presented in Table VIII reveal that tryptamine-NAD complex was not acted upon by NADglycohydrolase. In this respect A. niger NAD-glycohydrolase has a similar specificity as that of animal NAD-glycohydrolase. TABLE VIII EFFECT
OF
TRYPTAMINE oN NAD-GLYCOHYDROLASE
2/*moles of N A D and t r y p t a m i n e at the concentrations mentioned above were incubated for I h at 25 ° and t h e n o.i ml of the enzyme p r e p a r a t i o n was added, NAD-glycohydrolase was assayed b y measuring the a b s o r p t i o n of the cyanide complex at 34 ° m/*. Addition
None T r y p t a m i n e (2/*moles) T r y p t a m i n e (4/*moles)
Enzyme activity (ml*moleslh) 785 664 372
Inhibition (%)
-15 53
From the above results it is evident that the following characteristic groupings in NAD molecule are essential for NAD-glycohydrolase to act upon: (a) complete molecule of NAD, (b) quarternary nitrogen of nicotinamide molecule, (c) benzenoid structure of nicotinamide molecule, (d) free C-4 of nicotinamide, (e) free 6-amino group of adenine 16.
Biological role of NAD-glycohydrolase and nicotinamide amidase in the regulation of cellular NAD-concentration The object of purifying these two enzymes has been to study the substrate specificity and understand the biological significance of these two enzymes. The results presented in this communication revealed that both the enzymes, nicotinamide Biochim. Biophys. Aeta, 81 (1964) 311-322
ENZYMES OF N A D METABOLISM IN A .
niger
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amidase and NAD-glycohydrolase of A. niger are specific towards their respective substrates. The stoichiometry of the enzyme reactions was also established. Further methylation of nicotinamide and amidation of nicotinic acid could not be demonstrated in this mold 21. In view of the presence of nicotinic acid pathway of NAD synthesis 8, it was suggested that nicotinamide amidase plays an important role in deamidating nicotinamide to nicotinic acid, which can be reutilized for NAD synthesis. A potent biological source for nicotinamide would thus be obligatory for the operation of such a p a t h w a y in the cell. In the absence of amidation of nicotinic acid to nicotinamide 21, the only biological source in this organism appears to be b y the action of NADglycohydrolase on NAD. Further this mold exhibited the presence of both nicotinamide amidase and NAD-glycohydrolase activities. The presence of these two highly specific enzymes occurring together and the absence of other metabolic pathways for nicotinamide in this organism support the suggested cyclic pathway of NAD synthesis 8, according to which the liberated nicotinamide by the action of NAD-glycohydrolase on NAD gets deamidated to nicotinic acid which could be reutilized for NAD synthesis. In yeast JOSHI AND HANDLER7 have reported that NAD at and above certain concentration inhibits nicotinamide amidase. Thus when the concentration of cellular NAD increased it inhibited the nicotinamide amidase and thereby arrested the NADsynthesis from nicotinamide via nicotinic acid. Since the microbial NAD-glycohydrolases such as NAD-glycohydrolase of yeast is not inhibited by nicotinamide, the NADglycohydrolase will act upon the NAD and liberate nicotinamide, with a corresponding decrease in the level of cellular NAD. When the level of cellular NAD goes below the critical concentration, at which it can no longer inhibit nicotinamide amidase, then the liberated nicotinamide is reutilized for NAD synthesis via nicotinic acid, untilit reached a concentration sufficient for inhibiting the nicotinamide amidase. However, using purified preparation of nicotinamide amidase from A. niger it was observed that NAD at a level of 20 mg did not inhibit this enzyme activity to any great extent. Since nicotinamide at higher concentration was toxic to the growth of the mold 21, such a feedback mechanism as suggested by JosHI AND HANDLER~ in yeast m a y not be favourable to the mold A. niger. Probably NAD might exert a feedback control on nicotinic acid mononucleotide pyrophosphorylase (nicotinic acid mononucleotide: pyrophosphate phosphoribosyltransferase) rather than on nicotinamide amidase, an enzyme in the salvage p a t h w a y of NAD synthesis. ACKNOWLEDGEMENTS
Financial aid to D. S. R. SARMAand S. RAJALAKSHMIin the form of Senior Research Fellowships by the Council of Scientific and Industrial Research, New Delhi, is gratefully acknowledged. REFERENCES 1 D. E. HUGHES AND D. H. WILLIAMSON, Biochem. J., 55 (I953) 85 I. * Y. OEA, J. Biochem. Tokyo, 41 (1954) 89. 8 y . S. HALPERN AND H. GROSSOWlCZ, Biochem. J., 65 (1957) 716. * K. V. RAJAGOPALAN, D. S. R. SARMA, T. K. SUNDARAM AND P. S. SARMA, Enzymologia, 21 (1959) 277. 5 K. V. RAJAGOPALAN, T. K. SUNDARAM AND P. S. SARMA, Nature, 184 (1959) 461. 6 D. S. R. SARMA, S. RAJALAKSHMI AND P. S. SARMA, Biochem. Biophys. Res. Commun., 6 (1961) 389 •
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