On the mechanism of inhibiton of glutamate dehydrogenase by NADH and NADPH

342

BIOCHIMICA ET BIOPHYSICA ACTA

Preliminary Notes I'N (~IOO2

On the mechanism of inhibiton of glutamate by N A D H and N A D P H

dehydrogenase

It has been reported that NADH has two actions (>n glutamate dehydrogenase (Lglutamate :NAD (P)oxidoreductase (deaminating), EC t.4.I.3). One, observed at low protein concentrations, is inhibition of the glutamate dehydrogenase reaction; and the second, seen at higher protein concentrations, is disaggregation of the enzyme into large subunitsL Because of the apparent correlation between these two effects, it was proposed that they have a common mechanism. That is, that the reduced pyridine nucleotide produces a configurational change in the enzyme subunits which both prevents their aggregation, and inhibits the glutamate dehydrogenase reaction".

500

%

400

E 300

? 2OO ,8

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2

3

4 5 6 ? B NADH ( MxlO 4 )

9

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12

Fig. z. N A D H i n h i b i t i o n of g l u t a m a t e d e h y d r o g e n a s e c a t a l y z e d r e d u c t i v e a m i n a t i o n of (~-ketoglutarate, ~-aminobutyrate, and pyruvate, Reaction mixtures contained: o.o3M Tris-HCl buffer (pH 7.75), 1.2 . l o - ~ M E D T A , o.i M NH4C1, o.i M NaC1; a n d : o.o025 M (z-ketoglutarate a n d o.o94 m g / m l e n z y m e in 0 - ° - 0 ; o.o5 M a - k e t o b u t y r a t e a n d o . i o m g / m l e n z y m e in ~ - - A ; o.o25 M p y r u v a t e a n d I.I m g / m l e n z y m e in l - - l ; a n d N A D H as shown.

Recently, however, it was reported that the NADH-induced disaggregation of the enzyme required either divalent cations such as Zn 2+ (ref. 3) or certain other factors 4,a. Therefore, it became important to know whether N A D H can inhibit the catalytic activity of the enzyme under conditions where it has no detectable effect on enzyme aggregation. The experiments reported below show this to be the case, not only for the glutamate dehydrogenase reaction (~-ketoglutarate reduction), but also for the alanine- and c¢-aminobutyrate dehydrogenase reactions (pyruvate and c¢-ketobutyrate reduction). Tile addition of Z n 2+, however, which together with N A D H permits the Biochim. Biophys. Acla, 85 (I964) 342-345

343

P R E L I M I N A R Y NOTES G00

t l A t l l l

I l l l l

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Fig. 2. N A D P H i n h i b i t i o n of g l u t a m a t e d e h y d r o g e n a s e c a t a l y z e d r e d u c t i o n o f a m i n a t i o n o f cck e t o g l u t a r a t e , a - k e t o b u t y r a t e , a n d p y r u v a t e . E x p e r i m e n t p e r f o r m e d a s i n F i g . i e x c e p t for u s e o f N A D P H i n l i e u o f N A D H , a n d e n z y m e c o n c e n t r a t i o f l s of o.oo12 m g / m l i n O - - O , o . i o m g / m l in A--A, and i.i mg/ml in I--re.

enzyme to undergo the configurational change leading to disaggregation s, stimulated both the alanine- and a-aminobutyrate dehydrogenase reactions but inhibited the glutamate dehydrogenase reaction. Crystalline bovine glutamate dehydrogenase type I, NADH, and NADPH were purchased from the Sigma Chemical Company. The nucleotides were dissolved immediately prior to their use in order to avoid the inhibitor formation which sometimes occurs during storage of solutions ~. Enzyme assays were performed, as shown in the accompanying figures, by following the absorbancy change of the pyridine nucleotides at 34o m?~ during the first minute of the reaction. Light-scattering experiments were done with an Aminco light-scattering photometer as described previously 3. i

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1

Protein (mg/rnl) F i g . 3. E f f e c t o f N A D H o n l i g h t s c a t t e r i n g b y g l u t a m a t e d e h y d r o g e n a s e . R e a c t i o n m i x t u r e c o n t a i n e d 0.05 M T r i s - H C 1 b u f f e r ( p H 7.75), o . i M NaC1, o . I M NH4C1 , 1.2. i o - * M E D T A ; o.o25 M p y r u v a t e , e n z y m e c o n c e n t r a t i o n a s s h o w n , I . lO -4 M N A D H i n O ; a n d i • i o -s M N A D H i n Q.

Biochim. Biophys. Acta, 85 (I964) 3 4 2 - 3 4 5

344

PRELIMINARY N(I'I'ES

"file effects o f i n c r e a s i n g c o n c e n t r a t i o n s of N A I ) H on tin, ratc~ ,,I ~ ' d u c t i v ~ a m i n a t i o n o f a - k e t o g l u t a r a t e , ~ - k e t o b u t y r a t e , a n d p y r u v a t c arc s h o w u in Fig. t. T h e s e e x p e r i m e n t s w e r e d o n e in t h e p r e s e n c e o f 1.2. I o ~ M E I ) T A t(, ,,×~-Itl(l(' t h e p o s s i b l e effects o f m e t a l i m p u r i t i e s '~. I n h i b i t i o n o f t h e r e d u c t i , , n ()I: ( , - k e t o g h t t a r a t e , ~ - k e t o b u t y r a t e , a n d p y r u v a t e o c c u r r e d w i t h N A D H c o n c e n t r a t i o n s a b ( w , ' 4 ' Io ~ M, 6- IO -4 M, a n d 3" IO 4 M, r e s p e c t i v e l y . C o n c e n t r a t i o n s o f N A I ) P H a l ) . v c 3 " IO ~ 51 also i n h i b i t e d e a c h o f t h e t h r e e r e a c t i o n s (Fig. 2). L i g h t - s c a t t e r i n g e x p e r i m e n t s w e r e (tone t o c o n f i r m t h e f a c t t h a t n o disaggregati~ m o f t h e e n z y m e w a s p r o d u c e d b y tile p y r i d i n e n u c l e o t i d e s u n d e r c o n d i t i o n s of i n h i b i t i o n

08

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Fig. 4. Effect of Zn '2~ and NADH on reductive amination of ~-ketoglutarate and pyruvatc catalyzed by glutamate dehydrogenase. A, pyruvate reduction; B, a-ketoglutarate reduction. Experiments conducted as in Fig. I except for omission of EDTA and inclusion of t • io 4 M ZnCI2 in ~ - - ~ . Biochim. Biophys. Acla, t¢5 (I964) 342-345

PRELIMINARY NOTES

345

of the enzymic reactions. At an enzyme concentration of I.O mg/ml (the same as that used for the pyruvate amination experiments), neither N A D H nor NADPH, in concentrations as high as lO -3 M, lowered the weight average molecular weight of the enzyme. Furthermore, in the presence of all the reactants, neither nucleotide affected the concentration-dependent aggregation of the protein as shown by light scattering of the enzyme when measured as a function of enzyme concentration (Fig. 3). These data are consistent with those reported earlier showing that the reduced pyridine nucleotides alone had no effect on the weight average molecular weight in the absence of Zn ~+ (ref. 3). It m a y be concluded from these experiments that N A D H and N A D P H inhibit glutamate dehydrogenase-catalyzed reductive amination of these ketoacids under conditions where there are no changes in the structure of the enzyme which result in disaggregation. The experiments in Fig. 4 were performed without E D T A and with lO -4 M Zn ~+ to insure that the NADH concentrations used would actually lead to enzyme disaggregation. As shown, there was still enzyme inhibition when excess levels of N A D H were used, but the Zn 2+ stimulated the alanine dehydrogenase- (pyruvate reduction) while inhibiting the glutamate dehydrogenase-reaction (a-ketoglutarate reduction). In other experiments the reduction of a-ketobutyrate was also stimulated. These data are consistent with our previous findings that different configurations of glutamate dehydrogenase have different relative substrate specificitiesS, 7, that is, that the alanine (and apparently the a-aminobutyrate) dehydrogenase reaction is favored by configurational changes in the enzyme leading to its disaggregation. The same changes result in inhibition of the glutamate dehydrogenase reaction. The data also indicate that N A D H inhibition of the reactions is not dependent upon the same conformational changes in the enzyme as those leading to enzyme disaggregation and alanine dehydrogenase activation.

National Institute of A rthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Md. (U.S.A.)

K . LEMONE YIELDING GORDON M. TOMKINS DIANA S. TRUNDLE

1 C. FRIEDEN, J. Biol. Chem., 234 (1959) 809. C. FRIEDEN, J. Biol. Chem., 238 (I963) 3286. a K. L. YIELDING, G. M. TOMKINS AND D. TRUNDLE, Biochim. Biophys. Acta, 77 (1963) 703 • • K. L. YIELDING AND G. M. TOMKINS, Rec. Progr. Hormone Res., i8 (1961) 467 . s G. M. TOMKINS, K. L. YIELDING AND N. TALAL, Cold Spring Harbor Syrup. Quant. Biol., 28 (1963) 461. 6 C. P. FAWCETT, 1~. M. CIOTTI AND N. O. KAPLAN, Biochim. Biophys. Acta, 54 (1961) 21o. 7 G. ~¢[. TOMNINS, ~'{. L. YIELDING AND J. CURRAN, Proc. Natl. Acad, Sci. U.S., 47 (1961) 270.

Received February 24th , 1964 Biochim. Biophys. Acta, 85 (1964) 342-345