J. Mol. Biol. (1976) 100, 319-343

Studies of the Histidine Residues of Triose Phosphate Isomerase by Proton Magnetic Resonance and X-ray Crystallography C. A. BROWNE~, I. D. CA.MIPBELL~,P. A. KIENSRt, D. C. PHILLIPSw S. G. WA.LEY'~/kND I. A. WILSONw

Sir William Dunn School of Pathologyt Department of Biochemistry~ and Laboratory of Molecular Biophysics, Department of Zoology w University of Oxford South Parks Road, Oxford, England (Received 4 August 1975) A high resolution proton magnetic resonance study of triose phosphate isomerase (E.C. 5.3.1.1) from both rabbit and chicken is reported. The H el and H ~2 proton resonances of five histidines in the chicken enzyme, and one histidine in the rabbit enzyme, are observed to titrate in the pH range 5.4 to 9. The known amino acid sequences and the crystal structure determined by Banner eta/. (1975), have now been used to assign the resonances of histidine 100 in both enzymes and histidine 195 in the chicken enzyme. Details of the environments of the histidine residues are presented. I t is concluded that three conserved histidines, 95, 115 and 185 do not titrate in the pH range studied. Histidine 100 (and to a lesser extent histidine 195 in the chicken enzyme) is perturbed by the addition of ligands such as 2-phosphogiycollate, D-glyeerol-3-phosphate and dihydroxyacetone phosphate but is not perturbed by known inhibitors such as orthophosphate, pyrophosphate and phenyl phosphate. Members of the former set of ligands thus may bring about a change in conformation. Slowly exchanging peptide NH protons which resonate in the same region as the t t el and t t ~2 histidine resonances could be eliminated by unfolding the protein and refolding in 2H20. This procedure simplifies the spectrum and also shows that peptide NH resonances cover a large range and extend throughout the aromatic region. 1. I n t r o d u c t i o n Triose phosphate isomerase (E.C. 5.3.1.1), the enzyme in glycolysis that interconverts dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate, is now being much studied. Crystallographic studies started on rabbit TIMase and showed it to be a dimer of molecular weight about 53,009 (Johnson & Waley, 1967). The amino acid sequence has also been elucidated (Corrau & Waley, 1973,1974,1975). Further work, on crystals (Trentham et al., 1969) of chicken TIMase, has led to a complete three-dimensional structure (Banner et al., 1975) derived from interpretation of the 2.5 A resolution electron density map in the light of the sequences of the tryptic pcptides (Furth et al., 1974) and homology with rabbit TIMase. Meanwhile, detailed studies of the euergetics of the catalytic p a t h w a y are being carried out (Knowles t Abbreviations used: TIMase, trioso phosphate isomerase; n.m.r., nuclear magnetic resonance. 319

320

C. A. B R O W N E E T

AL.

et al., 1971). Spectroscopic studies have an important part to play in bridging the gap between crystallography and kinetics. I n particular, changes in conformation can often be observed and characteriscd in detail. This, in turn, opens the way to work relating changes in conformation to catalysis. The most suitable form of spectroscopy for detailed studies of the protein is n.m.r., because this teclmique offers the best chance of observing individual residues. Recent advances in technique enable proteins of molecular weight about 50,000 to be studied, although there has not been much work yet on enzymes containing subunits. The work to be described has concentrated on histidine resonances in rabbit and chicken isomerase; some progress has been made in assigning the resonances to individual residues, and towards characterising the change in conformation when ligands bind. This progress is a consequence of knowing the structure of chicken TIMase (Fig. 1) from crystallographic analysis (Banner et al., 1975). Comparison of rabbit and chicken isomerase has been helpful ; there are four residues of histidine in rabbit and eight in chicken, and their positions in the sequences are shown below. There is a high degree of homology, about 85%, between the rabbit and chicken enzymes. Chicken TIMase Rabbit TIMase

26

95 95

100 100

115 115

185 185

195

224

248

Positions of the histidine residues; the polypeptide chain of rabbit TIMase has 248 residues; a consistent numbering scheme is adopted by counting residue 3 of chicken TIMase as a deletion (Banner et al., 1975).

FIe. 1. The distribution of the eight histidine residues in one subunit of chicken TIMase. The course of the main polypeptide chain is shown by the connected a-carbon positions viewed in the direction of the non-crystallographic twofold axis (represented by the conventional syrabol) which relates the subunits. The histidine side-chains are shown in bolder lines and the position of the sulphate ion in the probable active site is marked S.

TRIOSE

PHOSPHATE

ISOMERASE

321

2. Materials and Methods (a) Materials Chicken and rabbit TIMase were prepared in the E n z y m e Preparation Laboratory (Oxford E n z y m e Group) under the direction of Dr M. P. Esnouf, chicken isomerase being prepared by the method of McVittie et al. (1972) and t h a t from rabbit by an unpublished method devised by Dr Esnouf. Sodium Dn-glycerol-3-phosphate, sodium r~-glycerol-3phosphate, 2-phosphoglycollate, and dihydroxyacetone phosphate were from Sigma (London) Chemical Co., Kingston-upon-Thames, SmTey, U.K. The tlScyclohexylamine salt of 2-phosphoglycollic acid in 2H20 was converted into the free acid with Dowex 50 (H + form), and the solution neutralized with deuterated NaHCOa.

(b) Preparation of samples Suspensions of protein in aqueous (NH4)2SO4 were dialysed against 5 mM-NH4HCO 3 containing a trace of dithiothreitol ; when the sulphate ions had been removed, the solution was dialysed against 5 mM-NH4HCO3 and freeze-dried. I f the initial suspension was only about 1 mg/ml in protein, it was first dialysed against 5 lm'~-NH4HC03 and then the solution concentrated by ultrafiltration ~4th an Amicon membrane before freeze-drying. Freeze-drying from too dilute a solution gave much denatured material. The dried enzyme was chilled and dissolved in cooled 2H20 containing 0.2 ~a-NaC1 and 20 rn~I-triethanolamine hych'ochloride; the concentration of protein was 50 to 100 mg/ml. The p H * was adjusted with NaO2H or 2HC1; p H * refers to the m et er reading in 2H20, the p H m et er having been calibrated with p H 4 and p H 9 buffers in water at 37~ (or at 45~ for the earlier n.m.r, work at this temperature). The p H * of each sample was measm'ed before and after collecting spectra and tim spectra were not used if the readings differed by more than 0.05. W h e n ligand was being added in small portions at a fixed pH, an ethylene diamine buffer was used. The enzymes were assayed, as previously described (Waley, 1973), before and after the n.m.r, experiments. (c) n.m.r, measurements The n.m.r, spectra were observed at 270 MHz on a Bruker spectrometer with an Oxford I n s t r u m e n t Co. superconducting magnet. The spectra shown in the Figures were obtained by collecting 2000 transients in 4096 data points (125 ~s/point). A Fourier transformation was then performed on 8192 points after applying a filter of line-width 2.5 Hz. Differences between spectra, and other data manipulations, were achieved as described previously by Campbell et al. (1974). The t em p era tu r e of the sample was 37~ (unless otherwise stated), and the chemical shift is given as parts per million (p.p.m.) dowlffield from sodium 2,2-dimethyl-2-silapentane-5-sulphonate. (d) Estimation of pK~ Tbe measured chemical shift (8) of a H e~ (or H ~2) histidine proton~ is, under conditions of fast exchange, the weighted mean of the chemical shifts of the unprotonated (SB) and protonated (6HS) forms. I f the amounts of these forms are B and H B , respectively, the total amotmt is B t , so t h a t the two equations :

B HB 1 = B---~.+ B t can be solved for B / B t and I-IB/Bt (King & Roberts, 1971). Division then gives B

HB

-

~ - ~HS 6s- 6 -

--

K~ [H+] "

-

-

(i)

The histidine protons are identified here by the abbreviations recommended by the IUPACIUB Commission on Biochemical Nomenclature. In the convention more commonly adopted in discussions of n.m.r, spectra H ~ and H 62 are denoted, respectively, the C(2~ and C(4) protons.

322

(3. A. B R O W N E

ET

AL.

I f we define the total change on protonation (span) as Zlo, i.e. Ao = ~8 -- $H~, a n d the change at a n y stage, reckoned from the high p H form as z1(A =- ~B -- 8), then K~ [H + ]

~o--

Useful alternative forms of this equation are: Zlo

zl

-

Ka -

~

[H+]

1

(2)

and ,to 1 -{- g a / [ H + ]

(3)

Eisenthal & Cornish-Bowden (1974) have given a convenient method for evaluating K~ and zJo in situations where the data take the form of equation (2). The "direct linear plot" obtained b y their method is illustrated in Fig. 11 ; the spread of cross-over points gives a measure of the confidence t h a t one can attach to the values of 1to and Ka obtained. This type of plot clearly reveals the interdependence of the parameters Ao a n d Ka. Once d o and Ka have been evaluated b y this method it is simple to generate a theoretical curve to fit the data b y the use of equation (3) (Fig. 4, Figs 7 to 10). The values of ,1o and Ka obtained from these plots are summarized in Table 1. A n i m p o r t a n t practical point is that triose phosphate isomerase (like most oligomerie proteins) is unstable below p H 5, and so a complete titration curve cannot be carried out. This means t h a t the mid-point, which is a convenient empirical measure of the pKa, cannot be located. Although the change in chemical shift of H el on ionization (do) is about 0.9 p.p.m. (Bradbury & Scheraga, 1966) it seems unwise to take this as fixed for any titrating histidine. A n approximate value can be found from the m a x i m u m slope of the titration curve, which is equal to --0.576 Zlo. The above procedure makes it relatively easy to see if the Henderson-Hasselbalch equation (1) is obeyed; addition of an electrostatic interaction factor does n o t improve the fit of histidine titration curves i n proteins, where this has been tried (Meadows, 1972). Marked deviations from the Henderson-ttasselbalch equation m a y be of structural significance (Cohen et al., 1973).

3. R e s u l t s

(a) Recognition of histidine resonances T y p i c a l 270 M/-Iz n.m.r, spectra of t h e chicken a n d r a b b i t e n z y m e s are s h o w n i n F i g u r e 2; t h e y are b r o a d l y similar, b u t there are detailed differences. The p a r t of t h e s p e c t r u m t h a t we are a t p r e s e n t concerned with is the a r o m a t i c region, l y i n g b e t w e e n 6 a n d 9-5 p.p.m., a n d i n p a r t i c u l a r the He 1 p r o t o n s of the lfistidine residues. These are f o u n d down~ield of t h e m a i n a r o m a t i c envelope, a t a b o u t 7-2 to 8.7 p.p.m., a n d are m a i n l y recognized b y their do~wafield shift of u p to 1 p.p.m, as the imidazole ring becomes p r o t o n a t e d . The area a n d l i n e - w i d t h are also aids to a s s i g n m e n t . W e can, a t the m o m e n t , o n l y recognize t h e histidine residues t h a t t i t r a t e ; moreover, we c a n n o t lower the p H * below a b o u t 5.3, a n d so a histidine with a p K below 4.9 would go u n r e c o g n i z e d ; t h e same also applies to one with a p K a b o v e 9. Our observations, then, centre o n histidines t i t r a t i n g w i t h i n these limits. (b) Rabbit muscle triose phosphate isomerase Difference spectra, where the spectra a t different p H values are compared, are v a l u a b l e i n detecting histidine resonances. T h u s one t i t r a t i n g histidine is revealed in r a b b i t TI1Vfase (Fig. 4, T a b l e 1). This histidine resonance was responsive to t h e

@,I

"'2.

+

9

+

N

v

i-

6 9 + oo ~

P-1

~I r

~

ca ~ l +i. +_~

324

C.A.

B R O W N E E T .,4L.

(o)

I

I

I

I

I

I

I

I

1

I

(b)

\ I0

I 9

I 8

I 7

I 6

I .5

:3

I 2

I 0

I -I

p.p. rn.

FIG. 2. 270 MHz proton magnetic resonanco spectra of (a) chicken muscle and (b) rabbit muscle TIMase; the conch of protein was 2 mM (in subunits), in 0.2 ~-NaCI in 20 mM-triethanolamine hydrochloride (pH* 7.4) at 37~ presence of ligands; the competitive inhibitor glycerol-3-phosphate (Burton & Waley, 1968) raised the p K to 6.2 (Ao ---- 0.82 p.p.m.) (Figs 3 and 4). The corresponding H02 proton resonance of this histidine could be detected above p H 6.5. The change in the chemical shift of the Her resonance was followed (at constant pH*) as glycerol-3-phosphate was added (Fig. 5(a)). The linear Scatehard plot (Fig. 5(b)) shows t h a t there are two binding sites per molecule of dimer, acting apparently independently. The value of the dissociation constant was 1-2 mM (in

pHm 6.3 pH e

Difference

I

1

I

I

I

I

II

I0

9

8

"7

6

p.p.m

FIo. 3. Aromatic region of the spectrum of rabbit muscle TIMase at p H * 6.3, and at p H * 7.2, a n d the difference between them.

~.

%,,

8.0

\~

~'~ t

I

6

t

I

7 pH~

t

I

8

Fze. 4. Titration data for the resonances in the histidine region of rabbit muscle TIMase. - - Q - - O - - , Unliganded; - - C ) - - O - in the presence of 13.3 mM-nr.-glycerol-3-phosphate. The points show experimental values, and the lines are theoretical curves, calculated as described in the text, with the parameters given in Table 1.

326

C. A . B R O W N E 2.0

ET

AL. 1.2

(a)

X

)'8

E/. 1.6

-

~

1.2

)'6

(o

.~ 0.8 E

)'4

0"4

)'2

c

c 4=

r

0

(b)

I I I 2 4 6 Total conca of n-qlycerol-3-phosphate

8

0

I 0.2

0"4

0'6

0"8

I0

(raM)

Fzo. 5. (a) Change of chemical shift ($) of the titrating histidine resonance of rabbit muscle TIMase with concentration of D-glycerol-3-phosphate. The conch of protein was 2 mM (in subunits, Mr 26,600) in 20 mM-ethylenediamine hydrochloride buffer (pH*6) containing 0-2 M-NaC1. The points are experimental, and the curve is the calculated one for a dissociation constant of 1.2 mM and concn of binding sites, 2 raM. (b) Plot of r/Irree against r, where r = $/$tot~], the fractional change in chemical shift of the titrating histidine resonance, and If tee is the conch of unbound D-glycerol-3-phosphate. 2H20 a t p H * 6.1, a t 37~ s o m e w h a t larger t h a n t h e v a l u e of a b o u t 0.5 mM f o u n d from kinetics, or e q u i l i b r i u m dialysis, u n d e r r a t h e r different conditions. W e believe, for reasons g i v e n below, t h a t t h e r e s o n a n c e which h a s j u s t b e e n discussed, is to be assigned t o H i s l 0 0 . T h e o t h e r t h r e e h i s t i d i n e residues (95, 115 a n d 185), then, do n o t t i t r a t e . (c) Chicken muscle triose phosphate isomerase T h e r e are e i g h t residues o f h i s t i d i n e in t h e chicken enzyme, a n d e x a m i n a t i o n o f t h e a r o m a t i c region o f t h e s p e c t r u m a t different p H v a l u e s (Fig. 6) reveals five t i t r a t i n g h i s t i d i n e resonances. T h e r e were no m a r k e d d e v i a t i o n s f r o m H e n d e r s o n H a s s e l b a l c h t i t r a t i o n curves (Fig. 7). T h e c a l c u l a t e d v a l u e s o f p K a a n d /1 o are given in T a b l e 1. One of t h e five t i t r a t i n g h i s t i d i n e resonances e in F i g u r e 7 is c l e a r l y set a p a r t from t h e r e s t a n d m u s t be in a special e n v i r o n m e n t ; t h e p r o b l e m o f assigning t h e residues is discussed below. R e s o n a n c e s a t t r i b u t a b l e t o Ho2 p r o t o n s could be p i c k e d out, a n d t h e i r t i t r a t i o n curves were, b r o a d l y , s i m i l a r to t h o s e o f t h e He1 p r o t o n s (Fig. 8). R e s o n a n c e a could n o t be followed t h r o u g h below p H * 7 ; t h e t i t r a t i o n curve for resonance e suggests i n t e r a c t i o n w i t h a n o t h e r g r o u p a t p H * a b o v e 7. W e now t u r n t o t h e effects o f ligands. T h e o n l y r e s o n a n c e t o be m u c h affected was resonance a; its t i t r a t i o n curve has " m o v e d a c r o s s " (Fig. 9). T h e c a l c u l a t e d curve h a d p K a 6"0 (Ao 1"09 p.p.m.). This is precisely t h e s a m e effect as was seen w i t h r a b b i t T I M a s e (Fig. 4). Moreover, b o t h resonances h a v e t h e s a m e shift (8.12 p.p.m.) a t pH*6. This r e s o n a n c e was also s e l e c t i v e l y b r o a d e n e d a n d so could n o t be d i s c e r n e d below pH*6. These effects were o b s e r v e d w i t h r a c e m i e glycerol-3p h o s p h a t e ; s e p a r a t e e x p e r i m e n t s s h o w e d t h a t L - g l y e e r o l - 3 - p h o s p h a t e h a d no effect on t h e n.m.r, s p e c t r u m , a n d so t h e changes discussed a b o v e are d u e t o t h e b i n d i n g o f t h e D-enantiomer, w h i c h is t h e i s o m e r o f t h e s a m e c o n f i g u r a t i o n as t h e s u b s t r a t e D-glyceraldehyde-3-phosphate. T h e effects of a n o t h e r i n h i b i t o r , 2 - p h o s p h o g l y c o l l a t e , were also e x a m i n e d . 2p h o s p h o g l y c o l l a t e h a s a lower K l (from kinetics) t h a n o t h e r i n h i b i t o r s o f T I M a s e

TRIOSE

PHOSPHATE

ISOMERASE

327

b c

pH*

6.7

6.9

7-3

7.45

7.7

7.9 I I0

I 9

I 8

I 7

I 6

p.p.m

Fie. 6. Aromatic region of the spectrum of chicken muscle TIMase at various pH* values revealing pH-dependent shifts. The resonances assigned to the histidine H el and H 62 protons are shown; the latter have the lower chemical shifts.

(Wolfenden, 1970); t h e value is similar to the dissociation c o n s t a n t for ])-glyceraldeh y d e - 3 - p h o s p h a t e (Webb & Knowles, 1974). Resonance a was again t h e one affected as is shown b y titration curves in the presence of 2-phosphoglycollate (Fig. 10) a n d a "direct linear p l o t " for resonance a in the absence a n d presence o f ligand (Fig. 11). Neither p y r o p h o s p h a t e nor p h e n y l p h o s p h a t e affected resonance a, a l t h o u g h b o t h appear to be competitive inhibitors (S. G. Waley, unpublished data).

328

C.A.

BROWNE

Jg~' A L .

8.5

8-0 ci

7.5

I

6

I

I

7

I

I

8

pH*

I

Fxo. 7. T i t r a t i o n d a t a for t h e r e s o n a n c e s (a to e) a s s i g n e d to h i s t i d i n e ]~E1 in t h e a r o m a t i c r e g i o n of t h e s p e c t r a of u n ] i g a n d e d c h i c k e n T I M a s e . T h e lines a r e t h e o r e t i c a l c u r v e s .

7.25

6"25

60-

~ ,

5"75- I 6

I

I 7

o

I pH*

] ~ ~ 1

~9

8

FIG. 8. Til~ration d a t a for t h e r e s o n a n c e s a s s i g n e d to h i s t i d i n o H ~2 o f u n l i g a n d e d c h i c k e n T I M a s e . T h e lines a r e t h e o r e t i c a l c u r v e s .

TRIOSE

PHOSPHATE

ISOMERASE

329

8.5

~176 9 ~

r

o\

~. 8-0 -

~o

\

~'o w~o

\

9 7.5

In

I

6

J

I

7 pH9

L

I 8

i

F I e . 9. T i t r a t i o n d a t a for c h i c k e n T I M a s o in t h e p r e s e n c e o f 13.3 m M - D L - g l y c e r o l - 3 - p h o s p h a t e . T h e lines a r e t h e o r e t i c a l c u r v e s .

Effects of substrate (dihydroxyacetone phosphate) on resonance a were observed, but measurements were precluded b y decomposition of the triose phosphates at 37~ in the presence of the isomerase. The products were mainly methylglyoxal and orthophosphate. This topic is being studied further. The effects of ligands on resonance e were relatively small; there was a small upfield shift of both the H~I and H~2 resonances at pH*6. The change is illustrated b y a plot of the difference in shift of the He1 resonance e with and without 2-phosphoglycollate at various p H values (Fig. 12), the values being derived from the calculated curves in Figures 7 and 10. (d) Peptide ~YH The large (unresolved) peaks at about 8.5 to 10 p.p.m, in Figure 2 are ascribed to the protons of peptide groups, i~Iany of these do not exchange with solvent 9H20 even after several hours at 37~ or 45~ Values for the numbers of unexchanged peptide H atoms after about 30 minutes at pH* 7.45, 37~ in 0.2 ~-NaC1, were calculated b y the method of H v i d t & Pedersen (1974). The n.m.r, spectra were divided into three areas; area C, 11.5 to 5.75 p.p.m., Lncludes both the peptide H atoms and the aromatic ones; area B, 5.75 to 3.5 p.p.m., includes the ~-Ctt protons, the fl-CH protons of serine and threonine, and the water signal; area A, 3.5 to --2 p.p.m, includes all other aliphatie protons ( B a k e t al., 1967). The number of aliphatie protons (a), of aromatic protons (c), and of peptide t I atoms (n) per subunit were calculated from the sequence. Then the number (N) of unexchanged peptide

330

C. A .

BROWNE

ET

AL.

@\

,,\. b.,o~

8.o

\ 7.5

*'X~ D

:,

I 6

a

I 7

I

\ /~176

e* ,

8

pH* Fza. 10. Titration data for chicken T I M ~ e in the presence of 10 mM-2-phosphog]ycoLLato. The lines are theoretical curves.

protons is given b y : N ---- C a / A - - c. The calculated values for rabbit were: a = 1063, c = 93, n - - 237, and the measured areas were A -~ 1.6225 and G ---- 0.261, whence N ---- 78. The calculated values for chicken were a ~-- 1037, c = 101, n ----240, and the measured areas were A ---- 1.498 and C =- 0.26, whence N ---- 79. These values, of about 80 uuexchanged peptide H atoms, m a y be compared with the results from tritium-hydrogen exchange (Browne & Waley, 1974), which showed t h a t about 270 H atoms/dimer had exchanged in after 30 minutes, and about 430 H atoms]dimer after 96 hours. The difference is 160 H atoms/dimer, or 80/monomer, as found from the n.m.r. ; this agreement m a y be held to suggest t h a t complete exchange (within experimental error) did indeed take place in 96 hours, but cannot be held to substantiate it. Since peptide N H protons are only prevented from rapid exchange by the noncovalent interactions in the folded molecule, these H atoms can be shed b y unfolding the protein and refolding in 2I-I20 (Waley, 1973; McVittie et al., 1972) containing some dithiothreitol; refolding occurred when the solution was diluted sixfold with 2H20. The solution was then dialysed against 5 mM-NH4HCO~ in 2H20 and freeze-dried. The n.m.r, spectrum of the 6 to 10 p.p,m, region was strikingly simplified (Fig. 13), and the histidine H ~z resonances stand out well. The difference spectrum (Fig. 13) shows t h a t the N H envelope covers a large range and extends through the aromatic region. The high-field region of the difference spectrum (not shown here) was featureless, which (together with the recovery of enzyme activity) showed t h a t the refolded protein had essentially the native conformation.

TRIOSE P H O S P H A T E

ISOMERASE

331

//j/ /

I0

t,.',/

0-75 0.

/

// // / / .c

/

/ / /

/

/

./

/

/

//

/

/

/

ii

/ / /

'.' 0 . 5 0

/

c

g

/

//

o

/

5

/

//

/,

/

/

/

/ / / /

/

2t___ -3

/

/

/

/

/

/

/ /

//

/

// //

/

/

,,./ 0.25

/ /

/

/

/

/

/

/ / /

/

-2

-I

0

I

106x[H +] F r o . 11. " D i r e c t linear plot" (Eisenthal & C o r n i s h - B o w d e n , 1974) of the H ~1 p r o t o n resonance a d a t a w i t h (. . . . . ) and without ( ) 10 mM-2-phosphoglyeollate. Pairs of m e a s u r e m e n t s of zl on t h e ordinate a n d [ H + ]* on the abscissa are joined b y lines ;/1 = 8B-8, w h e r e SB is the chemical shift (in p.p.m.) of the resonance at high p H * , a n d ~ is the chemical shift at an i n t e r m e d i a t e p H * .

A c c o r d i n g to e q u a t i o n (2) (see text) the intersections of the lines give values of/1 o (the total c h a n g e in chemical shift on protonation) on the ordinate a n d K a on the abscissa. T h e e x p e r i m e n t a l points are g i v e n in Figs 7 a n d 10.

4. D i s c u s s i o n (a) Assigning the histidine residues The environments of the eight histidine residues in chicken TIMase as seen in the crystal structure (Banner et al., 1975) are described in Table 2 and stereodiagrams are shown in Figures 14 to 21. The crystals were studied in 3.1 M-ammonium sulphate, at pH 7.4 (close to the isoelectric point of 7.27) and at 20~ The electron density map has a nominal resolution of 2-5/~ and provides independent images of the two subunits in the enzyme molecule. The subunits are related by an approximate twofold axis of s y m m e t r y but they differ in some details of their conformations as seen in the crystals (see Table 2 and text). These differences arise presumably because the subunits have different environments in the crystal structure and they m a y be taken to indicate some of the local variations in conformation that are accessible to the molecule in solution. The main chain and side chains of the enzyme molecule were fitted to the observed electron density as closely as possible by eye, but, given the limited resolution at this

2

332

C.A.

BROWNE

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FIG. 12. Variation with pH of change in chemical shift of the H ~1proton resonance brough~ about by the presence of 10 m~-2-phosphoglycollate. The curves shown are derived from the theoretical curves given in Figs 7 and 10. stage of the analysis, this does not always define the conformation of individual amino acid residues within close hmits. In particular for the histidine residues, it is often not possible to determine the conformational angle 2/2 (defining rotation about the fl-y bond) very closely and it is only possible to distinguish between X2 and )/2 -k 180 ~ from the electron density alone when the density corresponding to the nitrogen atoms is less well resolved from neighbouring density (representing hydrogenbonded groups) than that corresponding to the carbon atoms. The nature of the evidence from the electron density for the two independent subunits is noted in Table 2 in terms of the peak electron densities observed for the imidazoles (as a multiple of the root-mean-square error in the map) and the general shapes of the peaks. As is usual in such analyses, evidence for the orientation of an imidazole is often provided b y its environment especially when the nitrogen atoms can be placed at the proper distance from other polar atoms to form hydrogen bonds. This evidence is also listed in Table 2: it is most helpful when the interactions are with atoms t h a t have been well located (e.g. within helices) but interactions with probable water molecules represented in the electron density b y significant peaks in plausible positions, are also listed. Figures 14 to 21, which show only one conformation for each histidine (in one subunit), should be studied alongside the additional data in Table 2. The environments of the histidine residues are quite varied in nature, though 6 of the 8 are found in ~-hehces. Discussion here is restricted to the aspects that m a y be of value in interpreting the n.m.r, results. We proceed towards correlating individual resonances with individual residues by first comparing TIMase from rabbit and chicken, and then utilizing the information

TRIOSE

PHOSPHATE

ISONIERASE

333

o-b

I0

9

8

7

6

p.p.m

FIG. 13. Aromatic region of the spectrum of chicken TIMase, p H * 6-65, 45~ a with ~ protons; b with the N H protons exchanged for deuterium a t o m s ; a -- b the difference revealing the total broad N H proton band. The N H protons were exchanged b y unfolding the protein with 3 ~ - g u a n i d / n u m chloride in 2H20 a t p H 6-5, diluting sixfold with 2H20 a n d dialysing repeatedly against 5 m ~ - a m m o n i u m hydrogen carbonate, with a trace of dithiothreitol in ~H20, until no chloride could be detected in the diffusate; the solution was then freeze-dried.

summarized in Table 2. The argument can most easily be followed by reference to the scheme given below. As pointed out in the introduction, of the eight histidine residues of chicken TIMase, four occupy corresponding positions in rabbit. This set of four residues is discussed first. Since three out of the four residues in rabbit are non-titrating, our first task is to see ff this can be understood. The basis for this understanding will be that the three non-titrating histidine residues of chicken correspond to the three non-titrating residues of rabbit and, moreover, that analysis of the environments of

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FIa. 14. Environment of His95.2. In this and the following Figures the environments of the histidine residues in chicken TIMase are shown in detail. The numbers identify also the individual subunits (cf. Table 2). All atoms within 8 A of the histidine y-carbon are included. Presumed hydrogen bonds are indicated by broken lines. Residues shown in the thinner lines are from the adjacent subunit. I n rabbit, Iie92 is replaced by Va192.

Fie. 15. Environment of His185.2. All residues shown are conserved in rabbit TIMase.

these histidines as seen i n t h e crystal s t r u c t u r e of chicken T I M a s e applies also to r a b b i t TIMase. This is p a r t i c u l a r l y likely to be v a l i d for His95 a n d H i s l 0 0 , which occur i n highly conserved regions of t h e s t r u c t u r e , a n d for His185 which is b u r i e d i n a c e n t r a l n o n - p o l a r region. The small differences i n t h e e n v i r o n m e n t of H i s l l 5 (see :Fig. 17) seem u n l i k e l y t o affect its properties greatly.

TRIOSE PHOSPHATE

9

ISOMERASE

337

IIIS~

FIG. 16. Environment of Hisl00.2. All residues shown are conserved in rabbit TIMase. The side chain adopts different conformations in the two subunits (see Table 2 and text) in the crystal structure: in the one shown it is clearly exposed to solvent.

I

Fro. 17. Environment of Hisll5.2. I n rabbit TIMase, Pro70 is replaced by Thr70, Ala81 by Gly81 and Alall8 by SerllS. The side chain adopts different conformations in the two subunits in the crystal structure (see Table 2 and text), but in each it lies in a cleft in the surface of the enzyme.

(1) Histidine 95. The h y d r o g e n b o n d b e t w e e n t h e p e p t i d e N H of residue 97 a n d t h e n i t r o g e n a t o m N~I of His95 (Fig. 14) w o u l d have to be d i s r u p t e d for p r o t o n a t i o n of t h e histidine. I f t h e loss i n free energy o n d i s r u p t i o n of this b o n d were greater t h a n 2-5 to 3 keal/mol, t h e p K a of t h e histidine would be displaced to a v a l u e too low for

338

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FIo. 18. E n v i r o n m e n t of His195.2. This residue is on the surface of the molecule and interacts closely with Trpl91, though slightly differently in the two subunits (see Table 2 and text). The imidazole and indole rings are 2.7 to 3.0 A apart. Resonance e with pK~ 7.0 is assigned to His195.

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FIG. 19. E n v i r o n m e n t of His26.2.

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/ Fro. 20. E n v i r o n m e n t of His224.2.

TRIOSE

PHOSPHATE

339

ISOMERASE

Eight histidine residues

I

I

I

Five titrate

I-

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I

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Usual position

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l

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26 UR

95 NT

100 R

115 NT

185 NT

95

100

115

185

195 UP

224 UR

248 UR

Scheme for assigning histidine residues protonation to be observed in these experiments. Thus it seems reasonable to suppose t h a t His95 does not titrate, at ]east under our conditions. This residue is near Glu165, which from chemical evidence (Rose & O'Connell, 1969; Hartman, 1971; Miller & Waley, 1971; de la Mare et al., 1972) is in the active site. (2) Histidine 185. This residue is internal and the ring is surrounded b y nonpolar contacts (Fig. 15). This, then, is the second non-titrating histidine. Thus we can identify two non-titrating histidines with some confdence, but we need a third. This must be either Hisl00 or H i s l l 5 and, in making the choice, we can take into account the facts t h a t the titrating histidine with which we shall be

L~

FIG. 21. Environment of His248.2. This is the carboxyl-terminal residue. 23

$40

C.A.

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left in rabbit T1Mase is affected by ligands and that resonance a of chicken TIMase is also responsive, rather than unresponsive, to ligands. (3) Histidine 100. This residue takes up different conformations in the two subunits (Table 2); they are related by rotation about the ~-fl bond (Xa). The imidazole is fully exposed to solvent in one subunit (Fig. 16) and apparently interacts weakly, through a bifurcated hydrogen bond, with a carbonyl oxygen on the surface of the other. I t is close to, though not in direct contact with, Glu165. Hisl00 is, therefore, a strong candidate for identification as the single titrating histidine of rabbit TIMase and for the assignment of resonance a of chicken T l ~ a s e . (4) Histidine l l g . This residue also appears to take up different conformations in the two subunits (Table 2) : they are related mainly by rotation about the fl-7 bond (X2). In each subunit the imidazole ring hes in a cleft in the enzyme surface that is largely non-polar in character. There are many good non-polar contacts especially with Pro70 and Pro80 (in chicken, see Fig. 17). In one subunit, N(H)~I is close to the O(H) ~ of Glu119 though not in an appropriate orientation for hydrogen-bond formation. The N(H)~I and O(H)~ appear to interact directly with a common water molecule. In this subunit (Fig. 17), N(H)e2 is buried between Pro70 and Pro80 and is not involved in any good polar interactions. This environment, in which both nitrogens are not effectively solvated, might be expected to inhibit ionization of the imidazole, despite the proximity of the presumably charged Glull9. In the other subunit the imidazole appears to be arranged in such a way t h a t N(H)~2 approaches the O(H) E of Glull9, though again in an impossible orientation for hydrogen-bond formation, and N(H)6~ is exposed to solvent. This environment is not so clearly likely to inhibit ionization of the imidazole, though the non-polar contacts with the prolines (in chicken) are preserved. There is no evidence in either subunit that the residue adopts a conformation in which X1 is about -60 ~ and N(H)~I interacts weakly with the carbonyl oxygen of G l n l l l , although this conformation appears to be accessible. This description perhaps serves to illustrate the difficulty of predicting the ionization properties of protein side chains from their environments and to indicate the need for more work on this subject (e.g. Tanford & Roxby, 1972). On balance it may be reasonable to suppose t h a t His115 does not titrate in the pH* range 5-4 to 9 because of the non-polar elements in its preferred environment, but we must note that this is only a tentative conclusion. The adoption of different conformations in the two subunits also reminds us that there are conceivable circumstances in which a titrating histidine could give rise to such a broad reasonance that it would not be observed (Campbell et al., 1974). On the basis of these arguments our present conclusion is that resonance a of chicken and the single titrating histidine of rabbit TIMase should be assigned to ttisl00. We come now to discuss the histidine residues that are present only in chicken TIMase. (5) Histidine 195. This residue is close to Trp191 ; the rings are nearly, but not quite, parallel (Table 2 and Fig. 18). A marked ring-current effect on the chemical shift of the H~I and H~2 resonances will result from this arrangement, as discussed in detail below. Since none of the other histidine residues is in a comparable position, the assignment of resonance e, which is shifted up-field, to His195 seems especially firm.

TRIOSE PHOSPHATE ISOMERASE

341

(6) Histidines 26, 224 and 248. These residues are all surface or external (Table 2 and Figs 19, 20, 21) and resonances b, c and d are assigned to them. Although there is no firm evidence to say which corresponds to which, the possible interaction of His26 with Lys54 (Table 2) perhaps indicates t h a t resonance e (the lowest pKa of the three, Table 1) should be assigned to His26. This should be regarded as a v e r y tentative assignment. (b) Status of ring-current calculations The positions of resonances arising from atoms in a globular protein are affected by a variety of local perturbations. For a carbon-bound proton, however, the largest of these are due to ring-current effect of aromatic groups. These ring-current shifts provide not only a means of assignment, as with His195 in the present work, but also, in principle at least, a means of obtaining structural information. So far, however, this method has not been conspicuously successful; see, for example, a discussion of ring-current calculations for lysozyme (Campbell et al., 1975a). The v e r y specific interaction between His195 and Trpl91 (Fig. 18) potentially provides a good system with which to check ring-current calculations since the imidazole ring has two observable protons which are a fixed distance apart. I n considering the resonances arising from this residue in relation to its environment we m u s t note, however, t h a t the two chemically identical subuuits differ slightly in conformation in this region though in each the preferred orientation for the imidazole ring would seem to be with N~IH making a bifurcated hydrogen bond with the carbonyl oxygen of Trpl91 in the preceding t u r n of the helix in which both residues are found (Table 2, Fig. 18). As noted above it is likely t h a t these small eonformational differences arise from the different crystalline environments of the two subuuits: there is no evidence t h a t t h e y differ in solution. A fundamental problem in the calculation of ring-current shifts is the choice of a suitable reference compound to establish the zero. Here L-histidine was chosen. Table 3 then shows the results of calculations using two different sets of shift tables TABL~ 3

Observed and calculated shifts of histidine 195 Proton

Haigh-Malliont A1 A2

H~l H~2 H~ll]l H~12

0.549 0.23 0.23 1.047

0-474 0.40 0.491 0.521

Johnson-Bovey~f A1 As w 1.66 0.62 0.65 3.01

1-42 1.05 1-47 1.48

Mean 1.02 0.578 0.711 1-52

Observed:~ low pH high pH 1.24 1.22 1.24 1.22

0.96 1.48 0.96 1.48

t The ring current shifts from tryptophan 191 calculated using the tables of Halgh & Mallion (1972) and Johnson & Bovey (1958). The indole and benzcnoid parts of the tryptophan ring were treated separately with the assumption that the benzenoid part contributed 1.04, and the indole 0.56, times the contribution of a benzene ring (Giessner-Prettre & Pullman, 1965). :~ The observed upfield chemical shift (in p.p.m.) from the position observed by Sachs eta/. (1971) for L-histidine. wThe position of His195 with respect to Trpl91 is different in the two monomers in the X-ray model. The Table gives calculated shifts for monomers Ai and A2. II H6i and H ~2 correspond to the proton positions in the present model. Hall and H#ls correspond to the present (N)H~s and (N)H~i proton positions, respectively.

342

C. A. BROWNE E T A L .

that have been published (Haigh & Mallion, 1972; Johnson & Bovey, 1958). I t may be observed that the theories predict shifts which differ by as much as a factor of three but they agree in giving approximately the same H61/H ~2 shift ratio. Shifts predicted for the two subunits also differ by more than a factor of two. Column 6 of the Table gives the mean of the shifts predicted by the two theories for the two subunits. The mean predicted shifts for the two resonances with the imidazole rotated 180 ~ from the orientation shown in Figure 18 (He1' and H~2' in Table 3) correspond rather well with the observed shifts at lfigh p H values, but in view of the large variations in columns 2 to 5 it is doubtful if there is any justification for reversing the present orientation of the imidazole in the model. Another possibility is that the ring flips between two orientations, as has been observed for tyrosine residues in lysozyme (Campbell et al., 1975b). The mean shifts would then be predicted as 0.87 p.p.m, for H el and 1.05 p.p.m, for H~2.

(c) Ligand binding and its consequences The effects of inlfibitors on the titrating histidine resonances fall into two classes : (1) no effect observed: orthophosphate, pyrophosphate, L-glycerol-3-phosphate, phenyl phosphate. (2) Alteration of resonance a (and to a much lesser extent, resonance e): 2-phosphoglycollate, D-glycerol-3-phosphate; dihydroxyacetone phosphate. Ligands in class (2) alter the ultraviolet absorption (Johnson & Wolfenden, 1970) as does alkylation by bromohydroxyaeetone phosphate (de la Mare et al., 1972). Binding of orthophosphate, a class (1) ligand, does not have these effects. These observations help to distinguish between the (extreme) alternative consequences of a ligand binding to a macromolecule, namely charge effects and conformation changes. Thus the effects of the ligands in class (2) on the titrating histidine are probably not just a consequence of binding an anion to a rigid framework, because the two structurally different ligands have the same effect; moreover the (anion) binding of the class (1) inhibitors has no effect. What seems likely is that there is change in conformation when the class (2) ligands bind, which alters the position of Hisl00 and thus its pKa; the change in free energy of ionisation (from the change in pKa) is only about 0-6 kcal/mol (i.e. about 12% of the change in free energy when ligand binds). Another sensitive monitor of conformation is provided b y the close contact between the aromatic rings of His195 and Trpl91, and this registers a change in the shift of resonance e on ligand binding (Fig. 11). I t is interesting that this signal comes from a part of the structure that is well removed from the active-site region. Moreover, ligands have a marked effect on the conformational mobility of the protein (Browne & Waley, 1974), although here there is no comparable distinction between class (1) and class (2) ligands. Stabilization by ligands has also been shown b y their decreasing the rate of thermal inactivation (Wolfenden, 1970; P. H. Corran & S. G. Waley, unpublished data). The general conclusion from these observations is that when ligands bind, the structure tightens. Changes that seem quite small in geometrical terms (movements of say 0.5 A) may Well have marked effects on properties, especially functioning as a catalyst, and our results suggest that a combination of crystallographic and spectroscopic studies may enable us to define such changes more precisely than would be possible b y the use of either technique alone.

TRIOSE

PHOSPHATE

ISOMERASE

343

W e are grateful to the Medical Research Council, to the Science Research Council a n d to Dr M. P. Esnouf for his elaboration of p r e p a r a t i v e methods and guidance of the E n z y m e P r e p a r a t i o n Laboratory. This is a contribution from the Oxford E n z y m e Group. REFERENCES Bak, B., Pedel~en, E. J. & Sunby, F. (1967). J. Biol. Chem. 242, 2637-2645. Banner, D. W., Bloomer, A. C., Petsko, G. A., Phillips, D. C., Pogson, C. I. & Wilson, I. A. (1975). Nature (London), 255, 609-614. B r a d b u r y , J. H. & Scheraga, H. A. (1966). J. Amer. Chem. Soc. 88, 4240-4246. Browne, C. A. & Waley, S. G. (1974). Biochem. J. 141, 753-760. Burton, P. M. & Waley, S. G. (1968). Biochim. Biophys. Acta, 151, 714-715. Campbell, I. D., Linskog, S. & White, A. I. (1974). J. Mol. Biol. 90, 469-489. Campbell, I. D., Dobson, C. M. & Williams, R. J. P. (1975a}. Proc. Roy. Soc. set. A , 345, 41-59. Campbell, I. D., Dobson, C. M. & Williams, R. J. P. (1975b). Proc. Roy. Soc. set. B, 189, 503-509. Cohen, J. S., Griffin, J. H. & Scheehter, A. N. (1973). J. Biol. Chem. 248, 4305-4310. Corran, P. H. & Waley, S. G. (1973}. F E B S Letters, 30, 97-99. Corran, P. H. & Waley, S. G. (1974). Biochem. J. 139, 1-10. Corran, P. H. & Waley, S. G. (1975). Biochem. J. 145, 335-343. de la Mare, S., Coulson, A. F. W., Knowles, J. R., Priddle, J. D. & Offord, R. E. (1972). Biochem. J. 129, 321-331. Dickerson, R. E., Kendrew, J. C. & Strandberg, B. E. (1961). Acta Crystallogr. 14, 11881195. Eisenthal, R. & Cornish-Bowden, A. (1974). Biochem. J. 139, 715-720. F u r t h , A. J., Millman, J. D., Priddle, J. D. & Offord, R. E. (1974). Biochem. J. 139, 11-25. Giessner-Prettre, C. & Pullman, B. (1965). C.R.H. Acad. Sci. 261, 2512-2514. Haigh, C. W. & Mallion, R. B. (1972). Organic Mag. Res. 4, 203-228. H a r t m a n , F. C. (1971). Biochemistry, 10, 146-154. H v i d t , A. & Pedersen, E. J. (1974). Eur. J. Biochem. 48, 333-338. I U P A C - I U B Commission on Biochemical Nomenclature (1970). J. Mol. Biol. 52, 1-17. Johnson, C. E. & Bovey, F. A. (1958). J. Chem. Phys. 29, 1012-1014. Johnson, L. N. & Waley, S. G. (1967). J. Mol. Biol. 29, 321-322. Johnson, L. N. & Wolfenden, R. (1970). J. Mol. Biol. 47, 93-100. King, R. W. & Roberts, G. C. K. (1971). Biochemistry, 1O, 558-565. Knowles, J. R., Leadlay, P. F. & Maister, S. G. (1971). Cold Spring Harbor Syrup. Quant. Biol. 36, 157-164. MeVittie, J. D., Esnouf, M. P. & Peaeoeke, A. R. (1972). Eur. J. Biochem. 29, 67-73. Meadows, D. H. (1972). Methods in Enzymology, 26, 638-653. Miller, J. C. & Waley, S. G. (1971). Biochem. J. 123, 163-170. Rose, I. A. & O'Connell, E. L. (1969). J. Biol. Chem. 244, 6548-6550. Sachs, D. H., Scheehter, A. N. & Cohen, J. S. (1971). J. Biol. Chem. 246, 6576-6580. Tanford, C. & R o x b y , R. (1972). Biochemistry, 11, 2192-2198. Trentham, D. R., MeMurray, C. H. & Pogson, C. I. (1969). Biochem. J. 114, 19-24. Waley, S. G. (1973). Biochem. J. 135, 165-172. Webb, M. R. & Knowles, J. R. (1974). Biochem. J. 141,589-592. Wolfenden, R. (1970). Biochemistry, 9, 3404-3407.