Thermal stability of phosphoribosyladenosine triphosphate synthetase as reflected in its circular dichroism and activity properties. Effect of inhibitors

Biochimica et Biophysica Acta, 317 (1973) 123-13 ° © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - P r i n t e d in T h e N e t h e r l a n d s

BBA 36447 T H E R M A L S T A B I L I T Y OF P H O S P H O R I B O S Y L A D E N O S I N E T R I P H O S P H A T E S Y N T H E T A S E AS R E F L E C T E D IN ITS CIRCULAR D I C H R O I S M AND ACTIVITY P R O P E R T I E S . E F F E C T OF I N H I B I T O R S

H~KON

KRYV[

Department of Physiology, University of Bergen, N-5ooo Bergen (Norway) (Received J a n u a r y 25th, 1973)

SUMMARY

I. The circular dichroism (CD) of phosphoribosyladenosine triphosphate: pyrophosphate phosphoribosyltransferase (phosphoribosyladenosine triphosphate synthetase) from Escherichia coli in the far ultravidet region has been investigated. The CD spectrum of the enzyme is characterized by CD bands centering at 223, 21o and I 9 3 n m , with [0]4 = - - 1 4 8 0 0 , --14000 and + 1 8 0 0 0 degrees.cm~.dmole -1, respectively. A negligible dichroism in the near ultraviolet region has been observed. The analysis of the CD spectra based upon the published model parameters for a-, fl- and random structures suggests the presence of approx. 33% ahelix, 20-30% fl-structure, with the remaining portion of the enzyme existing in an unordered conformation. Histidine and AMP, inhibitors of phosphoribosyladenosine triphosphate synthetase, were found to have little or no effect on the CD spectra of the enzyme. 2. The inhibitors histidine and AMP both protect the ordered structure against unfolding or thermodenaturation, with histidine being the more effective of the two. 3- Upon heating to about 50 °C for IO min the negative ellipticity at 223 nm decreased irreversibly to a few percent of the value for the native enzyme, whereas only a slight reduction in the catalytic activity was observed. Thus, unfolding in parts of the native structure need not correspondingly influence the catalytic capability of the enzyme. 4. Both histidine and AMP stabilized the enzyme with respect to thermal inactivation.

INTRODUCTION

The enzyme, phosphoribosyladenosine triphosphate synthetase, is inhibited by AMP and is feedback inhibited by histidine 1-3. Ultracentrifuge studies have shown that the enzyme occurs in different aggregate forms, one of which, the 8.9-S species (mol. wt approx. 200 ooo), is stabilized by these inhibitors.

I24

it. I
Previous studies 5,6 have shown t h a t elevation of tim t e n l p e r a t u r e increases the t e n d e n c y of the e n z y m e to be i n h i b i t e d at high e n z y m e concentration, p r e s u m a b l y b y s t a b i l i z a t i o n of the 8.9-S species. The p r e s e n t work is a s t u d y of the t h e r m o d e n a t u r a t i o n ()f t)hosphorib()syl adenosine t r i p h o s p h a t e s y n t h e t a s e a n d of the influence of histidine and A M P on tiffs process. The effects of t e m p e r a t u r e on the unfolding of the p r o t e i n are d e m o n s t r a t e d b y tim use ()f tim CI) technique and are con~pared with its effect on the c a t a l y t i c act i v i t y of the enzyme. MATERIALS

AND

METH()I)S

Enzyme pr@aration amt chemicals P h o s p h o r i b o s y l a d e n o s i n e t r i p h o s p l m t e s y n t h e t a s e was p r e p a r e d ff()m our Escherichia colt m u t a n t strain X - I as described previously, including dialysis and r e - p r e c i p i t a t i o n L The e n z y m e was dissolved in o.5 ml of IO mM imidazole buffer cont a i n i n g o.5 ml 2 - m e r c a p t o e t h a n o l per liter (referred to as Basal buffer). F u r t h e r dilution of the e n z y m e was done in Basal buffer plus o.I M NaC1. AMP, A T P , p h o s p h o r i b o s y l p y r o p h o s p h a t e a n d purified p y r o p h o s p h a t a s e were o b t a i n e d from Sigma Chemical Co., St. Louis, Mo., U.S.A. Tris, imidazole, 2 - m e r c a p t o e t h a n o l a n d histidine were p u r c h a s e d from Merck AG, D a r m s t a d t , G e r m a n y . The c o n c e n t r a t i o n of p r o t e i n in solution was d e t e r m i n e d h y the m e t h o d of K l u n g s o y r s. Circular dichroism CD m e a s u r e m e n t s were p e r f o r m e d with a Jasco C D / U V J - I o recording spectrop o l a r i m e t e r in a cell with a o . I - m m light p a t h . The i n s t r u m e n t records the difference in a b s o r b a n e e (/1A) at a n y w a v e l e n g t h for left a n d right circularly polarized light. The difference in m o l e c u l a r e x t i n c t i o n coefficients (tIE = E~, -- Erd was c a l c u l a t e d from the relationship 1.t

AE

....

where c is the c o n c e n t r a t i o n in lnoles/l a n d d is the [)ath length in centimeters. The molecular ellipticities, 10], were t h e n o b t a i n e d from the equatu)n"(1 •

.t5oo

[0] = 2.303 •

. II-

where the units are degrees, cm". dmole 1. Tile calculations were based on a molecular weight of 2o0 ooo. The e x p e r i m e n t s designed to m e a s u r e the changes in the far u l t r a v i o l e t CI) s p e c t r a at e l e v a t e d t e m p e r a t u r e s utilized a H e t o circulator a n d t h e r m o r e g u l a t o r . The t e m p e r a t u r e of the cell wall was m e a s u r e d with a t h e r m i s t o r . After each t e m p e r a t u r e a d j u s t m e n t , i o min were allowed for t h e r m a l e q u i l i b r i u m between the p r o t e i n solutions, cell wall a n d the circulant (water). Once a d j u s t e d , the t e m p e r a t u r e was m a i n t a i n e d within + o . 2 °C.

PHOSPHORIBOSYLADENOSINE TRIPHOSPHATE SYNTHETASE

125

Reliable, low-noise CD records were obtained by averaging 3-4 scans at o. 4 nm/s.

N 2 flushing was used throughout the recording of the spectra.

Kinetics The activity determinations were carried out in a Shimadzu MPS-5oL multipurpose spectrophotometer as described previously 5. RESULTS

CD of the native enzyme Fig. I shows the ultraviolet CD spectra of phosphoribosyladenosine triphosphate synthetase. The spectrum has been corrected for a minor absorption artifact derived from the Basal buffer in the region below 200 nm. The enzyme exhibits large ellipticities with negative CD bands centering at 223 and 21o nm and a positive band at 195 nm. The three bands have molecular ellipticities, [0], of --14 800, --14 ooo and + 1 8 ooo degrees.cm 2. dmole -1, respectively. These average values, based upon three different enzyme preparations, indicate the presence of helical organization. Comparison of our CD data in this far ultraviolet region with the polylysine data of Greenfield and Fasman 1° suggests the presence of approx. 33% u-helix and 20-30% fl-structure, with the remaining segment of the enzyme folded in an unordered or random form, bearing in mind all the uncertainty in these types of calculations 1°,11. In the aromatic absorption region only a negligible dichroism was found (E0~es0 approx. IO degrees.cm ~-dmole-1). Changes in optical properties of a protein after binding of allosteric effectors or other ligands are not uncommon 12. Significant effects on the CD spectrum upon binding of substrates or inhibitors both in the farla, 14 and near ~5 ultraviolet regions have been demonstrated.

20

e~

b ,~o

- 20 I

200

I

I

240

I

?, (nm)

I

280

Fig. I. The far u l t r a v i o l e t c i r c u l a r d i c h r o i s m s p e c t r a of n a t i v e p h o s p h o r i b o s y l a d e n o s i n e t r i p h o s p h a t e s y n t h e t a s e a t 3 ° °C. The e n z y m e was d i l u t e d in Ba s a l buffer p l u s NaC1 a n d t h e c o n c e n t r a t i o n used r a n g e d from 1. 5 to 2. 4 m g / m l . A cell of o.oi m m o p t i c a l p a t h l e n g t h w a s used.

z26

H. KRYVI

F o r p h o s p h o r i b o s y l a d e n o s i n e t r i p h o s p h a t e s y n t h e t a s e , the inhibitors histidine a n d A M P have been found to stabilize w h a t is a s s u m e d to be the hexameric form of the e n z y m e 4. In an a t t e m p t to d e t e c t c o n f o r m a t i o n a l effects of these ligands on the enzyme, CD s p e c t r a have been recorded in the presence of histidine (o. 4 raM), A M P (o.25 mM), a n d histidine plus A M P (o.4 a n d o.25 lnM, respectively). The ligands h a d essentially no effect on the CD s p e c t r a of the e n z y m e both in the far or near ultraviolet regions. The results o b t a i n e d with these inhibitors indicate t h a t inhibition p r o d u c e d m) gross changes in folding and chain c o n f o r m a t i o n of the enzvme. This is consistent with the findings of Blasi ct al. ]6 for the same e n z y m e from Salmonella (vphim.urh~m. B i n d i n g of the inhibitors did not result in a n y exposure of tim a r o m a t i c groups.

Effects of inhibitors on thermal unfolding Changes in p h o s p h o r i b o s y l a d e n o s i n e t r i p h o s p h a t e s y n t h e t a s e confl)rmation a c c o m p a n y i n g t h e r m o d e n a t u r a t i o n were s t u d i e d b y m e a s u r i n g the far u l t r a v i o l e t CD, in Basal buffer, with or w i t h o u t t h e inhibitors histidine a n d AMP. W h e n heated, the n e g a t i v e b a n d s in the far u l t r a v i o l e t region of the CI) spect r u m progressively lost i n t e n s i t y as shown in Fig. 2. 7~ = 2 2 3

?', = 2 5 0 n m

am

z~E

I

I

I

5

10

20

Time

(min)

I

I

30

35

Fig. 2. Copies of recorder tracings showing the decrease of ellipticities at 223 n m fi)r the e n z y m e at elevated t e m p e r a t u r e as a function of time. The protein concentration in this e x p e r i m e n t was t.84 mg/ml. Curve a, e n z y m e in the absence of a n y ligand, t e m p e r a t u r e 51 C ; Curve b, enzyme in the presence of 25 ° / * M AMP, t e m p e r a t u r e 51 °C; Curve c, enzyme in the presence of 40o pM histidine, t e m p e r a t u r e 54 'C. R e p r e s e n t a t i v e e x p e r i m e n t s are shown. For details see text.

The e x p e r i m e n t s were carried out as follows: The samples were p r e h e a t e d at the e l e v a t e d t e m p e r a t u r e for 2 min in a w a t e r b a t h a n d t h e n t r a n s f e r r e d to the cuv e t t e where its circular d i c h r o i s m at 25o n m was recorded for a n o t h e r 2 rain. The w a v e l e n g t h was t h e n set at 223 n m a n d t h e recorder d r u m s t a r t e d . The d i s a p p e a r a n c e of the n e g a t i v e e l l i p t i c i t y at 223 mn was followed whilst keeping the t e m p e r a t u r e constant. W e h a v e used the r e a d i n g at 25 ° n m as a reference value since no change was o b s e r v e d at this w a v e l e n g t h u n d e r the conditions of t h e e x p e r i m e n t s . This holds for t h e B a s a l buffer, the p r o t e i n when heated, a n d the p r o t e i n in the presence of the inhibitors. F u r t h e r , when heated, no change in the circular dichroism at 223 n m for inhibi t o r in Basal buffer w i t h o u t p r o t e i n has been found. No visible t u r b i d i t y a p p e a r e d d u r i n g the heat t r e a t m e n t .

PHOSPHORIBOSYLADENOSINE

TRIPHOSPHATE

SYNTHETASE

12 7

In the results presented in Fig. 2, less than 15% of the peak remained after 35 min of heating at 51 °C in the absence of the inhibitors, whereas about 60 and 90% remained in the presence of AMP and histidine, respectively. Apparently, the inhibitory ligands histidine and AMP in s o m e w a y preserve the protein structure at high temperatures. The extent of protection seems to be different for the two, with histidine being the m o s t effective.

Temperature effects on the folded structure and enzymic activity The diagram in Fig. 3 shows the effect on the CD reading at 223 n m when phosphoribosyladenosine triphosphate synthetase in Basal buffer was heated to different temperatures. After heat t r e a t m e n t the samples were cooled in ice and the e n z y m e activities were measured at 30 °C as described in Materials and Methods. As before, the difference between the readings at 223 n m and 250 nm was used as an indication of the amount of ordered structure in the protein. a

100 i • 0 ~J

u

(1 0

>

ili~ii~ i!i~ii

w

5O i~ii~i !ii

i li,iil,

b 45 Temperature

49 (°C)

Fig. 3. Effect of incubation of the enzyme at elevated temperatures on ta) the catalytic activity and (b) the size of the 223-nm peak. The enzyme solution (2. 4 mg/ml in Basal buffer) incubated for io min at a controlled temperature. Aliquots were taken for recording of the CD spectra. The rest of the sample was cooled in ice bath and activity measurements were performed at 3o °C. The measurements are compared to the values obtained for unheated enzyme at 3° °C which are taken as ioo%. In order to check the reversibility of the disappearance of ordered structure, the CD spectra were also recorded after cooling the samples. In samples heated to temperatures above 43 °C, no 223-nm peak reappeared after incubation for 24 h in ice. It should be m e n t i o n e d that the temperature at which the a c t i v i t y of the e n z y m e rapidly decreases is strongly dependent on the concentration of the protein in solution as shown in Fig. 4. This relatively strong dependence of denaturation

I28

H. K R Y V I C

0.4[-

so S 0.3

300

6

12

[Enzyme] (mg/ml)

C

•

0

~

. . . . . .

40

T i m e (min) 80

F i g . 4- T h e v a r i a t i o n o f h e a t i n a c t i v a t i o n o f p h o s p h o r i b o s y l a d e n o s i n e triph~sphatt~ s\'nthctase as a function of protein eoncentration. The ordinate represents the temperature at which the rate o f t h e c a t a l y t i c r e a c t i o n a t a n y c o n c e n t r a t i o n o f t h e e n z y m e w a s h a l f o f t h e r a t e o b t a i n e d a t 3 ° ('. Incubation time at every temperature step was 5 rain. Fig. 5. H e a t i n a c t i v a t i o n o f p h o s p h o r i b o s y l a d e n o s i n e t r i p h o s p h a t c s y n t h e t a s e a t 4,S ( . I n c u b a t i o n m i x t u r e s c o n t a i n e d 3 m g / m l e n z y m e . , \ l i q u o t s o f 2o p l w e r e t a k e n o u t a t t h e i n d i c a t e d t e m p e r a t u r e s a n d a s s a y e d i m m e d i a t e l y . "|'he c u r v e s r e p r e s e n t t h e loss in a c t i v i t y o n h e a t i n g o f t h e e n z y m e ( ( , ) in t h e p r e s e n c e o f I . - h i s t i d i n e ( 0 , 8 . 0 . 1 o a r a M ; II, 6 . 9 - 1 o ~ r a M ; &, 3 - 4 5 " J o 3 r a M : V , 6 . 9 " ~o a r a M ) . It is n o t e d t h a t t h e h i g h e s t c o n c e n t r a t i o n o f h i s t i d i n e i n f l u e n c e s t h e a s s a y .

t e m p e r a t u r e on enzyine c o n c e n t r a t i o n makes it difficult to m a t c h all the e x p e r i m e n t s of this kind to e x a c t l y the same t e m p e r a t u r e . Therefore, Fig .3 shows the results of one r e p r e s e n t a t i v e e x p e r i m e n t . In all the e x p e r i m e a t s , a lower t e m p e r a t u r e was required for the d e s t r u c t i o n of the 223-mn CD deflection than was r e q u i r e d for inactiv a t i o n of e n z y m e function. While 7o~o of the a c t i v i t y still r e m a i n e d at 4!J "C, ~mlv a b o u t 8% of the helix c o n t e n t was left. This m a k e s us suggest t h a t the " a c t i v e site" of the e n z y m e is more stable a g a i n s t heat d e n a t u r a t i o n t h a n is the helical portion of the enzyme.

Heat inactivatio~z and iJ~hibitors Several enzymes have been shown to be stabilized t o w a r d s heat i n a c t i v a t i o u b y their feedback i n h i b i t o r 1,17,1s. W i t h a p p r o p r i a t e c o n c e n t r a t i o n s of histidine Martin ~ has d e m o n s t r a t e d this effect for p h o s p h o r i b o s y l a d e n o s i n e t r i p h o s p h a t e p y r o p h o s p h o r y l a s e from S. typhimurium. H e found t h a t at low histidine c o n c e n t r a t i o n s the e n z y m e is stabilized to h e a t i n a c t i v a t i o n , whereas at higher c o n c e n t r a t i o n s the enzyme is r e n d e r e d more heat sensitive. Fig. 5 shows the b e h a v i o r of pt~osphoribosyladenosine t r i p h o s p h a t e s y n t h e t a s e froul E. coil to heat i n a c t i v a t i o n after i n c u b a t i o n with different c o n c e n t r a t i o n s of histidine. The result is a l m o s t the same as f o u n d b y M a r t i n ~. B u t although we h a v e found an o p t i m a l c o n c e n t r a t i o n of histidine for stabilization, some p r o t e c t i o n was o b s e r v e d even at high histidine c o n c e n t r a t i o n s . E x p e r i m e n t s were also carried out with A M P as the stabilizing ligand ([rig. 6). I t was found t h a t A M P stabilizes the e n z y m e to h e a t i n a c t i v a t i o n . The peculiar increase in r a t e at higher c o n c e n t r a t i o n s of A M P 5 to Io rain after the beginning of the h e a t t r e a t m e n t has been o b s e r v e d t h r o u g h o u t this series of e x p e r i m e n t s .

PHOSPHORIBOSYLADENOSINE TRIPHOSPHATE SYNTHETASE

129

1.0

0

O to

@

.> 0.5

@

J

0

i

i

I

,

r

,

40

--..i

80 Time(rnin)

Fig. 6. H e a t i n a c t i v a t i o n of p h o s p h o r i b o s y l a d e n o s i n e t r i p h o s p h a t e s y n t h e t a s e a t 47.5 °C in t h e presence of different c o n c e n t r a t i o n s of AMP. I n c u b a t i o n m i x t u r e s c o n t a i n e d 1.9 m g / m l e n z y m e . A l i q u o t s of 2o/,1 t a k e n o u t at i n d i c a t e d t e m p e r a t u r e s a n d a s s a y e d i m m e d i a t e l y . I n order to s t a n d a r d i z e t h e e x p e r i m e n t s t h e rate m e a s u r e d for u n h e a t e d e n z y m e a t 3 ° °C is t a k e n as I o o % . T h e c u r v e s r e p r e s e n t t h e loss in a c t i v i t y on h e a t i n g t h e e n z y m e (O) in t h e presence of 50 p M A M P (O), 5 °0/~M A M P (11) a n d 5 m M A M P (A).

DISCUSSION

The unfolding of ordered protein structure, or the transition of the a-helical part and fl-structure segments into randomly coiled structures, m a y be demonstrated by recording the circular dichroism in the far ultraviolet region of the spectrum. As the unfolding proceeds, a gradual reduction towards negative values of the peak at 193 nm is seen. At the same time, the negative peaks between 208 and 223 nm are abolished and become slightly positive 1°. Because of relatively poor resolution, and high absorbance from buffer and ligands at I95 nm, and the special sensitivity of the 2o8-nm peak to distortions 19,2°, we have chosen to follow the negative peak at 223 nm as a measure of the amount of native structure preserved in the enzyme. We believe that the scattering and flattening distortions found for membraneous suspensions and heavily aggregated protein systems discussed by several authorn, ~9-22 do not apply to our case. This is because: (i) Our instrument records simultaneously the circular dichroism phenomena and the photomultiplicator voltage which is proportional to the absorbance in the sample measured. Only a very minute increase in this voltage (0.02 V) was observed for all the samples referred to in Fig. 2 during the 35 min of recording. No differences were detectable between the samples. (2) As shown in Fig. 3, the activity is only slightly reduced when the 223-nm

I3O

H. KRYVI

peak is almost lost. Thus, the change in conformation must be relatively limited since a general change in structure would be expected to lead to loss of activity. Previous studies have shown that phosphoribosyladenosine triphosphate synthetase exists in a number of oligomeric forms with different sedimentation properties 4. Histidine and AMP stabilize the hexameric forms (8. 9 S) of the enzyme, and a combination of these ligands seems to lock the enzyme in a rigid, inactive form. On the basis of these findings a model was formulated. The results in Fig. 2 show that the conformational changes induced by histidine and AMP protect the ordered structure against thermodenaturation. Histidine and AMP also protect the enzyme against thermal inactivation (Figs 5 and 6). it is of great interest, however, that activity remains after the complete loss of helical structure. Previous studies have shown that histidine had no effect on the exchange ~f labeled hydrogen between protein and solvent ", AMP inhibited hydrogen exchange and histidine enhanced the AMP effect on the exchange of hydrogen. Apparently, phosphoribosyladenosine triphosphate synthetase possesses several sites and structures which are affected in different ways by the specific ligands and by heat. Some of these structures are necessary for the activity of the enzyme and its control, while others are not. ACKNOWLEDGEMENTS

I am grateful to Dr L. Klungs0yr for advice and stimulating discussions throughout this work, and to Mrs Trine Tydal for technical assistance.

REFERENCES I 2 3 4 5 6 7 8 9 io ii i2 13 14 I5 16 17 t8 19 20 2~ 22

Martin, R. G. (I963) J- lliol. Chem. 238, 257 208 Voll, M. J., Appella, E. a n d Martin, R. G. (I967) ,/. Biol. Chem. z42, 170o--I7o 7 K l u n g s o y r , L., H a g e m a n , J. H., Fall, L. a n d A t k i n s o n , D. E. (I968) Biochemistry 7, 4°35--4°4 ° K l u n g s o y r , L. a n d K r y v i , H. (1971) Biochim. Biophys. Acta 227, 327-336 K r y v i , H. a n d K l u n g s o y r , L. (I97 I) Biochim. Biophys. Acta 235, 429-434 K l u n g s o y r , L. (1971) Biochemistry Io, 4875-488o K l u n g s o y r , L. a n d A t k i n s o n , D. E. (197 o) Biochemislcv 9, 2o2i 2027 K l u n g s o y r , L. (1969) Anal. Biochem. 27, 91-98 I n s t r u c t i o n M a n u a l for J A S C O Model J - i o , pp. 2,2o Greenfield, N. a n d F a s m a n , G. D. (1969) Biochemistry 8, 4 r o 8 - 4 1 1 0 U r r y , D. W. (1972) Biochim. Biophys. Acta 265, 115-168 Gratzer, W. B. a n d Cowburn, D. A. (t969) Nature 222, 426 43 ~ D r a t z , E, A. a n d Calvin, M. (I966) Nature 211, 497-5Ol O h t a , T., S h i m a d a , I. a n d I m a h o r i , K. (1967) J. Mol. Biol. 26, 519 524 Sorensen, M. E. a n d H e r s k o v i t s , T. T. (i972) Biochim. Biophys. Acta 257, 2o--29 Blasi, F., Aloj, S. M. a n d Goldberger, R. F. (1971) Biochemislry io, 14o9--14I 7 C h a n g e u x , J. p. (1961) Cold Spring Harbor Syrup. Ouant. Biol. 26, 313 S t a d t m a n , E. R., Cohen, G. N., Le Bras, G. a n d de R o b i c h o n - S z u h n a j s t e r , lt. (L901) J. t~iol. Chem. 236, 2033-2038 Urry, D. "W. a n d Ji, T. H. (I968) Arch. Biochem. Biophys. 128, 8o2 8o 7 L i t m a n , B. J. (I972) BiochemistriJ II, 3243-3247 Steim, J. M. a n d Fleischer, S. (1967) Proc. Natl. Acad. Sci. U.S. 58 , t292 L298 Schneider, A. S., Schneider, M.-J. T. a n d I~osenheck, I~. (197 o) Proc. Nall. Acad. Sci. U.S. 66, 793-798