Aspartate transcarbamylase from Escherichia coli I. Inhibition by inorganic anions

45 °

i~i()(;m.xlr(:A ET BI()I'IiVSI(:A ACT\

ira:\ 05459 A S P A R T A T E TRANSCARBAMYLASE FROM Escherichia coli I. I N H I B I T I O N BY I N O R G A N I C ANIONS

KJ E L L K L E P P E *

Norsh Hydro's Institute Jor Cancer Research, Montebello, Oslo (Norway) (Received M a r c h i 6 t h , I966)

SUMMARY

I. The effect of different inorganic anions on the catalytic activity of native and subunit aspartate transcarbamylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2 ) has been investigated at pH 7.o and 25 °. 2. Several inorganic anions were found to inhibit both native and subunit aspartate transcarbamylase. The order of effectiveness fi)r the best inhibitors was: PPi > F - > Pi > SO423. For each ion except F , a strict competitive relationship was observed between the anion inhibitors and the substrate carbamyl phosphate. 4. The concentration of L-aspartate also greatly influenced the magnitude of the inhibition. The inhibition increased with increasing concentration of L-aspartate. 5" The effect of F - was shown to be due to a displacement of the p H curve along the p H axis. F inhibited native aspartate transcarbamylase below pH 8 and activated it above this pH. 6. Possible mechanisms of inhibition are discussed, and it is suggested that an inhibitor--L-aspartate complex is formed at the active site.

INTRODUCTION

Aspartate transcarbamylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli has within the last few years received increased interest due to its unique properties as a regulatory enzyme in the biosynthesis of the pyrimidines. Aspartate transcarbamylase was first demonstrated in E. coli by REICHARD AND HANSHOF~'I,2, who purified the enzyme approx, ioo-fold and studied some of its properties. SHEPHERDSON AND PARDEE 3 observed that the enzyme could be induced in large amounts in certain mutants of E. coli, and they succeeded in crystallizing it. More recent studies b y GERHART AND P A R D E E 4 - s and A b b r e v i a t i o n : PCMB, p - c h l o r o m e r c u r i b e n z o a t e . * Fellow of t h e N o r w e g i a n Cancer Society.

Biochim. Biophys. Acta, I22 (I966) 45 ° 461

ATCASE AND INORGANIC ANIONS

451

GERHART AND SCHACHMAN 9 have shown that native aspartate transcarbamylase is composed of several catalytic and regulatory subunits. These can be made to dissociate by treating the native enzyme with heat or reagents such as PCMB. Under certain conditions native aspartate transcarbamylase is inhibited by CTP and activated by ATP, whereas the monomeric enzyme is not affected by these nucleotides. It is thought that CTP and ATP may bind to specific sites on the regulatory subunits of native aspartate transcarbamylase, which are distinct from the active sites, or binding sites for the substrates. Aspartate transcarbamylase from E. coli has also lately been classified as an allosteric enzyme1°, 11. It displays a very complex type of kinetics, common to many of the regulatory enzymes. The present study, which was undertaken as a preliminary study to a more extensive investigation of aspartate transcarbamylase from E. coli, deals with the effect of moderate concentrations of inorganic anions on this enzyme. Very little information is available in the literature as to the effect of different salts and buffers on aspartate transcarbamylase. It seemed highly desirable to know more about these aspects since this might possibly throw light on the nature and mode of action of this enzyme. It appears from the present work that a number of inorganic anions will inhibit both native and subunit aspartate transcarbamylase. Mostly the inhibition is strictly competitive with the substrate earbamyl phosphate. The magnitude of the inhibition however also increases with increasing concentration of L-aspartate. A preliminary communication describing some of these findings has appeared 12.

MATERIALS

Chemicals Dilithium carbamyl phosphate and most of the nucleotides were obtained from Sigma Chemical Company, U.S.A. Carbamyl phosphate was recrystallized before use, according to the method of GERHART AND PARDEE5. CTP was from Mann Research Laboratories, U.S.A. Sodium pyrophosphate was a product of E. Merck, Germany. It was recrystallized from water before use. Imidazole was from Eastman Organic Chemicals, U.S.A. All other chemicals used throughout this study were of analytical grade, unless otherwise stated. Enzyme preparation Aspartate transcarbamylase was prepared according to the procedure of GERHART AND PARDEE5 from a mutant, R-I85-482, of E. coli, kindly provided by Dr. A. B. PARDEE, Princeton University, U.S.A. Subunit aspartate transcarbamylase was separated from native aspartate transcarbamylase b y passing the enzyme through a column of Sephadex G-2oo (90 cm × 1.5 cm). The native aspartate transcarbamylase thus obtained gave a single protein peak on centrifugation in a sucrose density gradient, suggesting a high degree of homogeneity of the enzyme sample. The specific activity was slightly higher than that reported by GERHART AND PARnEE 5, and was approx. 8000 units/mg protein. The native aspartate transcarbamylase was inhibited by CTP to approximately the same degree as previously reported 5. No inorganic pyrophosphatase activity was detected in the enzyme sample. Biochim. Biophys. Acta, 122 (1966) 45o-46I

452

K. KLFPPI:I

Catalytic subunit aspartate transcarbamylase was prepared by heating native aspartate transcarbamvlase according to the method of (~ERHART AND PAR1)I,;E7. METHODS

The activity of the enzyme was determined by measuring tile carbamyl aspartic acid formed by a method slightly modified from that of GERHAI~T AND PARDEE'5. After the addition of K2S208 the tubes were kept in the waterbath at 25 ° for exactly 25 min. They were then cooled to approx. 5 ° in a different water bath and read immediately afterwards at 56o m/~. Control experiments were carried out by measuring the amount of Pi formed in the reaction by the method of LowRy AND LOPEZ13. These gave essentially the same results as with the first method. Certain compounds, such as those that contained Br-, NO 3 , C10 a or S C N , interfered with the color formation in the first method; when this occurred the activity was determined solely by the second method. The effect of I-- could not be measured since it interfered with both assay methods. In general the reaction was run for IO min at 25 ° and the rate calculated. In most experiments it was found that this rate was equal to the initial rate. However, when low concentrations of carbamyl phosphate were used it was necessary to run a series of reactions for different periods of time and calculate the initial reaction rate from these. The buffer used in most experiments was o.o5 M imidazole-o.o3 M acetate -o.oooi M EDTA (pH 7.o). Imidazole and acetate do not haw~ an3' effect on the activity of aspartate transcarbamylase at these concentrations. Protein was determined by the method of LowRY et al. '4, and occasionally also by the method of KALCKAR~'~. The inhibitor constants were determined from Lineweaver-Burk plots 16, using the simple Michaelis-Menten relationship ~7, and also by the method of DIXON~8. These two methods gave essentially the same results. RESULTS

Inhibition by different anions In a previous communication '* it was shown that PPi was a very efficient inhibitor of aspartate transcarbamylase from E. coli. In the present work a number of other anions has also been tested for inhibition. Table I shows the °/o inhibition caused by various inorganic anions for native aspartate transcarbamylase at a fairly low concentration of carbamyl phosphate. Under the same conditions the inhibition of subunit aspartate transcarbamylase is approximately equal to that of native aspartate transcarbamylase. With the exception of F-, polyvalent anions are the best inhibitors of aspartate transcarbamylase. By far the most effective inhibitor is PPi, followed by F-, Pi and SO42-. For the anions belonging to the Hofmeister series, i.e. S Q 2-, CI-, NO3- and SCN- (refs. 28, 29), some resemblance exists between the original lyotropic series and the order of inhibiting effectiveness of these ions. Regarding the inhibition by the halides it should be noted that the sizes of the ionic radii for the halides are in the following order F < C I - < Br-(ref. 19), while Biochim. Biophys. Acta, 122 (I966) 45o-461

453

A T C A s E AND INORGANIC ANIONS TABLE I INHIBITION

OF NATIVE

ASPARTATE

TRANSCARBAMYLASE

BY" I N O R G A N I C

ANIONS

The reaction m i x t u r e s contained: O.Ol 5 M L-aspartate, o.8 mM c a r b a m y l p h o s p h a t e , 25 mM inhibitor and o.I #g protein in a t o t a l volume of 0. 5 ml. The buffer was: o.o 5 M imidazole-o.o 3 M a c e t a t e - o . o o o I M E D T A (pH 7.0). T e m p e r a t u r e 25 °. All the anions were added as sodium salts, and the solutions were a d j u s t e d to p H 7.0 b y addition of acetic acid or N a O H .

Inhibitor

Inhibition by 25 m M

(%)

HP207 zHPO42-

SO42HAsO42-

NO 3CIO:c SCNHCO sBrC1F-

91 35 3° 25 15 4 o o o lO 39

the magnitude of the inhibition caused b y these ions is exactly in the opposite order, i.e. the smallest ion gives the largest inhibition. P i is one of the products of the enzymatic reaction, and the possibility exists that the inhibition observed with P i might be due to a reversal of the reaction. This does not, however, appear to be so. Other experiments confirmed the earlier findings of REICHARD AND HANSHOFF2 that the enzymatic reaction for all practical purposes is irreversible. i

i

i

\

o

*QOSM Pi

•

Q04 _z •a ~ M P~ o3

Ld i~

0.02

i

I

i

2

,4

6

~/[C.ARB/~tYL PHOSPHATE](mlv~' Fig. I. Effect of c a r b a m y l p h o s p h a t e c o n c e n t r a t i o n on the inhibition of native a s p a r t a t e t r a n s c a r b a m y l a s e b y El. The reaction m i x t u r e s contained: O.Ol 5 M L-aspartate and o . I / ~ g protein in a total v o l u m e of o. 5 ml. The c o n c e n t r a t i o n of c a r b a m y l p h o s p h a t e and Pi varied as s h o w n above. The buffer was: 0.o 5 M imidazole-o.o3 M a c e t a t e - o . o o o I M E D T A (pH 7.0). T e m p e r a t u r e 25%

Biochim. Biophys. Acta, 122 (1966) 45o-461

454

K. I(LI':PI't: i

I

1

/0.03

_z ? u) I.u J 0

0.02

0.01

I

I

I

1

2

3

]/[CARBAMYLPHOSPHATE](ram)-I Fig. 2. Competitive inhibition of native a s p a r t a t e t r a n s c a r b a m y l a s e bv C1 . E x p e r i m e n t a l conditions as for Fig. t.

Effect of carbamyl phosphate concentration Evidence has previously been presented that PPi is a competitive inhibitor with carbamyl phosphate 12. The influence of different concentrations of carbamyl phosphate on the inhibition has now been investigated for all the anions listed in Table I, and both for native and subunit aspartate transcarbamylase. With all ions except F-, the inhibition decreases and can be fully abolished with increasing concentrations of carbamyl phosphate. Figs. i, 2 and 3 show Lineweaver-Burk plots for tile anions Pi, C1- and F-, with native aspartate transcarbamylase. With Pi and C1- straight lines are obtained which cross each other on the I/V axis, suggesting competitive inhibition with carbamyl phosphate. At the concentration of L-aspartate used in these experiments, O.Ol5 M, the apparent Ki for Pi and C1- was found to be 3" IO a M and Io 2 M respectively. A', for PPi under the same conditions was 8- lO _5 M, and Km for carbamyl phosphate 5 • lO-5 M. This value of Km for carbamyl phosphate 0.02

+0.025 M NaF :E z :E "T tD IM

+0.OI25M NaF

O.O1

CONTROL

d :E

A

I

I

I

1

2

3

,~CARBAMYLPHOSPHATE](mM)-' Fig. 3. N o n - c o m p e t i t i v e inhibition of native a s p a r t a t e t r a n s c a r b a m y l a s e by F - . E x p e r i m e n t a l conditions as in legend to Fig. i.

Biochim. Biophys..4cta, 122 (1966) 45o-461

ATCAsE AND INORGANICANIONS

455

is approx. Io-fold lower than t h a t determined b y REICHARD AND HANSHOFF2 under slightly different conditions. F - is unique among the inhibitors studied. In the presence of F - almost parallel lines are obtained when the results are plotted according to the method of LINEWEAVER AND BURK 16. Thus the inhibition by F - cannot be abolished by employing larger concentrations of carbamyl phosphate. Preincubation of asparrate transcarbamylase with F - had no effect on the magnitude of the inhibition observed. A number of compounds such as nucleoside-5'-di- and triphosphates contain PPi as an integral part of the molecule ; these nucoeltides might therefore be considered to be derivatives of PPi. As such they should also be expected to inhibit aspartate transcarbamylase in a manner similar to that found for PPi, but as shown in Table I I TABLE II INHIBITION

OF NATIVE

ASPARTATE

TRANSCARBAMYLASE

BY NUCLEOSIDE-5t-TRIPHOSPHATES

The e x p e r i m e n t a l c o n d i t i o n s were t h e s a m e as those d e s c r i b e d in t h e l e ge nd t o T a b l e I, e x c e p t t h a t t h e i n h i b i t o r c o n c e n t r a t i o n w a s I mM a n d t h a t of c a r b a m y l p h o s p h a t e 0.8 mM or 8 mM.

Inhibitor

ATP CTP GTP UTP PPt

Inhibition by r m M inhibitor (%) 0.8 m M carbamyl phosphate

8 m M carbamvl phosphate

o 26 9 2 5°

o 20 5 I II

for the nucleoside-5'-triphosphates , this does not appear to take place to any extent. Increasing concentrations of carbamyl phosphate do not affect the inhibition by these compounds significantly. CTP is the best nucleotide inhibitor, and GERHART AND PARDEE 5 have shown that inhibition caused b y this and other nucleotide inhibitors can be overcome by increasing the concentration of the substrate L-aspartate. They have also suggested that these nucleotides are bound to certain sites on the regulatory subunits of native aspartate transcarbamylase 7-9. Their findings regarding the competitive nature between L-aspartate and the nucleotides have been confirmed in this work. The effect of a number of phosphate esters, such as glucose 6-phosphate, ribose 5-phosphate, p-nitrophenylphosphate, and a number of nucleoside-5'-phosphates has also been investigated, but these compounds do not inhibit aspartate transcarbamylase under conditions similar to those used for the anions in Table I. The inability of these compounds and the nucleotides to inhibit aspartate transcarbamylase competitively with carbamyl phosphate m a y be related to their large size and bulkiness as compared with the substrate carbamyl phosphate. Inhibition b y PPi and P i has also been observed with aspartate transcarbamylase from other microorganisms. N E U M A N N AND J O N E S ~° have demonstrated that these compounds inhibit aspartate transcarbamylase from Pseudomonas fluorescens. PPi was found to be only partially competitive with carbamyl phosphate. However, Biochim. Biophys. Acta, 122 (1966) 45o-461

I,i. KI.I,:I'I'I:

4.%()

it is apparent that the most efficient nucleotide inhibitor ()f this aspartate transcarbamvlase, UTP, and Pt)i must bind to different sites ()n the enzyme'.

Fa[fect of L-aspartate co~zcentration The difference in the mechanism of inhibition observed between the nucleotides and PPi suggested the possibility that L-aspartate might also affect the inhibition by the various anions to some extent. The influence of different concentrations of Laspartate on the inhibition was therefore studied f()r some anions. Fig. 4 shows i

0.04

i

i

f

i

HEATED (CATALYTIC SL~BUNIT)

NA'i'IV E

+lmM

P~

i 0.02 :E::t

1

I

100 200

I

I

I

300 0 100 200 ~/[L-ASPARTATE]

I

300

Fig. 4. Effect of L-aspartate concentration on the inhibition of native and s u b u n i t a s p a r t a t e t r a n s c a r b a m y l a s e by PPi. The reaction m i x t u r e s contained: o.8 mM c a r b a m y l p h o s p h a t e , o.ooi M t)Pi, w h e n used, and o . i / , g protein in a total v o l u m e of o. 5 ml. The buffer was: o.o 5 M imidazole-o.o 3 M a c e t a t e - o . o o o I M E D T A (pH 7). T e m p e r a t u r e 25 °.

Lineweaver-Burk plots of saturation curves in the presence and absence of PPi for native and subunit aspartate transcarbamylase. In the absence of PPi the shape of the curve for the native enzyme resembles that of a parabola with the axis on the I/V axis, whereas for subunit aspartate transcarbamylase a straight line is obtained. This is in complete agreement with the findings of GERHART AND PARDEE7. l.Aspartate shows homotropic co-operative effects with native aspartate transcarbamylase, but not with subunit aspartate transcarbamylase. In the presence of inhibitor the curve for native aspartate transcarbamylase is unusual in form. It has a distinct minimum around o.o13 M L-aspartate. Higher concentrations of L-aspartate lead to a decrease in the rate, and thus an apparent inhibition by the substrate is observed in the presence of the inhibitor PPi. For subunit aspartate transearbamylase a straight line is obtained in the presence of PPi, which is almost parallel with the control. These lines do not cross on the I/S axis : inhibition by substrate is therefore observed in the presence of PPi for subunit aspartate transcarbamylase as well, but it is not as pronounced as for native aspartate transcarbamylase. Since the inhibition becomes greater with increasing concentrations of Laspartate the apparent K, for PPi will also decrease with increasing concentration of L-aspartate. This is illustrated in Fig. 5 both for native and subunit aspartate transcarbamylase. The decrease in K, is much greater for native aspartate transcarbamylase than for heat-treated aspartate transcarbamylase, but at high concentrations of L-aspartate the Ki for PPi is approximately the same for both enzyme species. The Biochim. Biophys. Acta, 122 (1966) 45o-461

457

ATCASE AND INORGANIC ANIONS

I

MOLARITY

2 OF"

3

L-ASPARTATE x10 2.

Fig. 5. Effect of L-aspartate concentration on the inhibitor c o n s t a n t of PPi for native and subunit a s p a r t a t e transcarbamylase, p H 7.0 and t e m p e r a t u r e 25 °.

shape of these curves bears some resemblance to the saturation curves found for enzymes with normal Michaelis-Menten kinetics. A plateau is reached at a certain concentration of the substrate. The importance of this finding with regard to the mechanism of inhibition bv the anions will be elaborated in more detail in the discussion. Similar results to those described for P P i have also been obtained with PiF-, which is non-competitive with carbamyl phosphate, also shows a similar type of effect with different concentrations of L-aspartate. Here, however, the inhibition by the substrate is more marked both for native and heat-treated aspartate transcarbamylase. This is illustrated in Fig. 6. It thus appears that the concentration of L-aspartate also deeply affects the magnitude of inhibition caused b y inorganic anions. In general the inhibition will be greatest at low concentrations of carbamyl phosphate and high concentrations of L-aspartate. I t is interesting to note that the effect of L-aspartate on the inhibition by anions is exactly opposite to that found for the inhibition by the nucleotides.

Effect of pH on the inhibition by PP, and FIn attempts to explain the inhibition observed with F - the effect of p H on the inhibition was studied for some inhibitors. Fig. 6 shows the effect of p H on the P P i 1

0,04

aoa ~

NATIVE

i

,

P

,

i

H~ATED

(~Tm,T~c~SUNiT)

z

::E ~1

0.02

Fig. 6. Effect of L-aspartate concentration on the inhibition of native a n d s u b u n i t a s p a r t a t e t r a n s c a r b a m y l a s e by F - . E x p e r i m e n t a l conditions as in legend to Fig. 4.

Biochim. Biophys. Acta, 122 (1966) 45o-461

45~S

K. K KEl'l'K

L9 150 ?" :E 100

8 50

7

pH

8

9

Fig. 7. Effect of p H on t h e i n h i b i t i o n of n a t i v e a s p a r t a t e t r a n s c a r b a m y l a s e by P P i a n d F . The r e a c t i o n m i x t u r e s c o n s i s t e d of: 0.8 mM c a r b a m y l p h o s p h a t e , o.o~ 5 M L - a s p a r t a t e , a n d o.1 /*g p r o t e i n in a t o t a l v o l u m e of o. 5 ml. The c o n c e n t r a t i o n of r ' P i a n d F - w a s as s how n above. The buffers were: p H 6.5--8.o, o.o5 M imidazole, o.oool M E D T A a n d v a r i o u s a m o u n t s of a c e t a t e ; p H 8.25 -9.25, o.o5 M Tris, o.oool M E D T A a n d v a r i o u s c o n c e n t r a t i o n s of a c e t a t e . T e m p e r a t u r e 25%

and F inhibition of native aspartate transcarbamylase. With PPi the pH optimum in the presence of this inhibitor is shifted about o.I pH unit to the right to pH 7.6. The extent of inhibition by PPi is slightly less on the alkaline side than on the acid side of the p H optimum. With F - the p H optimum is shifted farther to the right to about pH 7.95 : aspartate transcarbamylase is actually activated by F " at p H values higher than pH 8, and inhibited at lower p H values. The pH curves in the presence and absence of F-- are almost identical in form and height, differing only in position on the p H axis. Both sides of the velocity p H curve have been displaced about o.45 p H unit. The type of interaction displayed by F can therefore be explained in terms of changes produced by the inhibitor in the ionization constants of certain groups in the active site of the enzyme responsible for the pH curve. Similar types of interactions to that described above for F have been obserw~d for other enzymes with different types of anions. Fumarate hydratase (E(" 4.2.I.2) (ref. 21) is one example where a number of anions cause displacement of the velocity-pH curve along the ptl axis. DISCUSSION

The results from the present study demonstrate that a number of anions at moderate concentrations will inhibit aspartate transcarbamylase from E. coN. With all ions except F-, there is a strict competitive relationship between the anions and the substrate carbamyl phosphate. F-, on the other hand, competes with neither carbamyl phosphate nor l.-aspartate. Here the effect is on the p K of certain groups in the active site, leading to inhibition below pH 8 and activation above p H 8. The most efficient inorganic anion inhibitor, PPi, has a certain structural similarity to carbamyl phosphate, and this probably accounts for the rather high affinity observed with this ion. Although PPi and the other anions will undoubtedly bind to a number of different groups on the protein molecule, their inhibitory effect can best be explained b y assuming that these ions compete with carbamyl phosphate Biochim. Biophys..4eta, 122 (t966) 4 5 o - 4 6 t

A T C A S E AND INORGANIC ANIONS

459

for the carbamyl phosphate-binding sites. The effect of F - is, however, somewhat difficult to reconcile with this mechanism, but with the halides it may well be that the magnitude of the inhibition will depend on the charge density of the ions, while the type of inhibition will be determined by the size of the ion. The facts that Brdoes not inhibit, and that C1- is competitive with, and F - is non-competitive with carbamyl phosphate support this view. Thus F - may well bind to the same group as carbamyl phosphate, but since it is such a small molecule it will not affect the binding of carbamyl phosphate ; it will only influence the p K of the groups responsible for the pH curve. The inorganic anion inhibitors and carbamyl phosphate are all negatively charged, and thus the binding site for carbamyl phosphate must involve at least one basic group, possibly two. The rather small Km observed for carbamyl phosphate further suggests that the affinity of aspartate transcarbamylase for carbamyl phosphate is much higher than was previously thought. The observation that L-aspartate deeply influences the magnitude of the inhibition caused by the anions is interesting and deserves further comment. At the present time several possible explanations can be given for this phenomenon. It might be that L-aspartate changes the conformation or other properties of the protein in such a way that the apparent affinity of these inhibitors will increase. But in view of the fact that Kra for carbamyl phosphate is not significantly affected by the concentration of L-aspartate, this explanation seems unlikely. A more plausible explanation for this finding involves the idea that an inhibitor-L-aspartate complex is formed at the active site in the following way: EI

77 E

% EIA

EA

where E stands for one active site, I for inhibitor and A for L-aspartate. It is thought that the inhibitor will be bound more tightly to the enzyme in the ternary complex EIA than in the binary complex El, possibly due to the influence of the amino group on L-aspartic acid. It is not yet clear, however, whether or not the enzyme preferentially uses one of the pathways listed above for forming this complex. The difference between K, of PPi for native and subunit aspartate transcarbamylase in Fig. 5 then has a natural explanation. The large decrease in the apparent Ki found for native aspartate transcarbamylase with increasing concentrations of L-aspartate can be ascribed to the allosteric nature of this enzyme, i.e. the affinity of the enzyme for L-aspartate increases as the concentration of L-aspartate becomes greater. Thus the amount of EIA will also increase with increasing concentrations of L-aspartate. For subunit aspartate transcarbamylase the affinity of L-aspartate is independent of the concentration of this substrate; there is therefore a marked difference in inhibition between native and subunit aspartate transcarbamylase at low concentrations of L-aspartate, whereas at high concentrations of L-aspartate there is little difference in the magnitude of inhibition shown by the two enzyme species. For the same reason it is clear that the inhibition by substrate found in the presence of inhibitors will be more pronounced for native aspartate transcarbamylase than for subunit aspartate transcarbamylase. Biochim. Biophys. dcta, i22 (1966) 45o-461

400

K. KLEI'I'F

GERHART AND PAR I)EE 5 first showed t h a t n a t i v e aspartate transcarbamylas(. displays rather complex kinetics with respect to L-aspartate. The results f r , m th(, present s t u d y confirm a n d p a r t l y e x t e n d these observati(ms. However, (it~HaJ~r AND PARDEE used phosphate buffers in most of their studies a n d this fact to some e x t e n t complicates the i n t e r p r e t a t i o n of their results. W i t h respect to a n i o n effects on enzymes it seems r e l e v a n t also to m e n t i o n t h a t for some proteins it has recently been shown t h a t anions influence the t e r t i a r y or q u a t e r n a r y structure 22-'4, and the rate of association a n d dissociation of s u b u n i t s in such a process as cold i n a c t i v a t i o n of enzymes is also affected b y anions"'~,")6. Substrates m a y as a rule either accelerate or p r e v e n t such changes from t a k i n g place. I n the present studies no a t t e m p t has been made to correlate changes in the protein s t r u c t u r e with inhibition, a n d therefore the possibility t h a t anions affect the protein s t r u c t u r e c a n n o t completely be ignored. FRIDOVICHz7 has recently observed a n i o n i n h i b i t i o n of acetoacetate decarboxylase (EC 4.1.i.4) a n d he has proposed an interesting theory to explain the results. I t is t h o u g h t t h a t the i n h i b i t i o n b y anions depends on changes in the structure of water coincident to the formation of the enzyme a n i o n complex. It seems, however, doubtful whether this hypothesis can be applied to aspartate t r a n s c a r b a m y l a s e since FRIDOWCH found m a x i n m m i n h i b i t i o n with m o n o v a l e n t anions, whereas p o l y v a l e n t anions are the best inhibitors for aspartate transcarbamylase. The biological significance of the findings reported in the present s t u d y has been briefly discussed elsewhere '~. Both PPi a n d Pi are universally d i s t r i b u t e d in biological materials; it is therefore expected t h a t these ions will interfere with the a c t i v i t y of a s p a r t a t e t r a n s c a r b a m v l a s e in systems i n vivo, and hence they m a y play a role in cellular control mechanisms. In this respect the recent s t u d y of NEU.~IANX AND JONES 2° on e n d - p r o d u c t i n h i b i t i o n of various aspartate t r a n s c a r b a m y l a s e s is of importance, since it d e m o n s t r a t e s t h a t aspartate t r a n s c a r b a m v l a s e s from other organisms are also affected b y these ions.

ACKNOWLEDGEMENT The a u t h o r is grateful to Professor A. PIHL for helpful discussions and criticism of the m a n u s c r i p t .

REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13

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