Alkaline conformational transition of chicken egg white lysozyme

Biochimica et Biophysica Acta, 328 (1973) 31-34 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - Printed in The N e t h e r l a n d s

BBA

36548

A L K A L I N E CONFORMATIONAL T R A N S I T I O N OF C H I C K E N EGG W H I T E LYSOZYME

A. A. A B O D E R I N a, E. B O E D E F E L D b AND P. L. L U I S I b

aDepartment of Biochemistry, College of Medicine, University of Lagos, Lagos (Nigeria) and bTechnisch-Chemisches Laboratorium, ETH, Zi~rich (Switzerland) (Received April 24th, 1973)

SUMMARY

An enzymatically active derivative of chicken egg white lysozyme, 0ty r(4-nitrobenz-2-oxa-I,3-diazolyl)-lysozyme, undergoes a conformational transition in the alkaline region. The associated thermodynamic parameters (pKa = 9.89; A H = 4.74 kcal/mole; A S = --29.9 cal/°C'mole at 3oo °K) suggest the dependence of the transition on the ionization of a tyrosyl residue. The relevance of these observations to the native enzyme is discussed.

Different lines of recent evidence suggest that chicken egg white lysozyme undergoes a conformational transition at alkaline pH value close to the isoelectric point (pH II). (a) The mean residue rotation at 199 nm changes from + 1 6 800 ° at pH 7 to + 1 4 65 °o at p H 9.051. Similarly, there is a sharp positive increase in the ellipticity values going from p H 5.8 to alkaline p H values 2, even though no clear trend in the changes in ellipticity in the far ultraviolet region can be found between p H 9.1 and i i . (b) The fluorescence quantum yield of lysozyme shows a marked decrease at high pH. This feature has been attributed to the ionization of a tyrosyl hydroxyl group, as has the loss in rotatory power at 199 nm 1. We 3 have recently described the preparation of a derivative of lysozyme, v i z . : 0tyr-(4-nitrobenz-2-oxa-I,3-diazolyl)-lysozyme (0tyr-NBD-lysozyme), which is fully enzymatically active and with a conformation which shows only small differences to that of the native enzyme. Furthermore, the 0tyr-NBD chromophore is sensitive to the polarity of medium. For example, the absorption peak at 380 nm of the chromophore in the model compound 0tyr-NBD-N-acetyl-L-tyrosinamide (NATA) in water is shifted to 370 nm in ethanol. The spectral characteristics of the chromophore are independent of pH as it does not possess any ionizable group s. We report the use of this derivative of lysozyme, with its convenient reporter system, for the study of the nature of the conformational states of the native enzyme. A b b r e v i a t i o n s : NBD, 4-nitrobenz-2-oxa-i,3-diazolyl; NATA, Otyr-NBD-N-acetyl-L-tyro sinamide.

32

A. ABODERIN et al.

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115 105

99 95 O~

85

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240

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360

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Fig. i. Ultraviolet s p e c t r u m of Otyr-NBD-lysozyme as a function of pH. Concentration of protein 1.o9" lO.5 M. Buffers where o.o 5 M; p H 2.3, citrate; p H 7.2 and 11. 5, p h o s p h a t e ; p H 8.5, 9.5, 9.9, lO.2 and lO. 5, borate. Temperature, 275 °K.

The spectrum of the chromophore on the protein as a function of pH is shown in Fig. I. This is a simple transition with a well-defined isosbestic point at 35o nm. A plot of the changes in absorbance either at 33o and 38o nm yields a typical titration curve which, at 275 °K, has a pKa of lO.13 ~ 0.03 (Fig. 2). The changes are reversible. The change in the spectrum of the protein-bound Otyr-NBD chromophore is most easily rationalized as involving a transfer of the chromophore from a wholly aqueous environment to a hydrophobic one. This blue shift in the spectrum (5o nm) 7

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pl-I Fig. 2. Plot of a b s o r b a n c e changes at 33 ° n m Temperature, 275 °K-

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) and 380 n m ( - - . - - ) as functions of p H .

33

CONFORMATIONAL TRANSITION OF LYSOZYME

10.1

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1/T × 10 3 deg -1 F i g . 3. v a n ' t H u f f p l o t f o r t h e v a r i a t i o n o f t h e p K a w i t h t e m p e r a t u r e . standard deviation.

The vertical bars indicate

is one of the largest to be observed in such chromophores which are sensitive to the polarity of the solvent medium. However, the insolubility of the model compound, NATA, in highly non-polar solvents such as benzene and cyclohexane makes it impossible to directly confirm this shift on the model compound. The variation of the pKa with temperature has been used to estimate both the enthalpy and the entropy changes associated with transition. A plot of the van 't Huff equation in Fig. 3 shows that within the limits of the accuracy of the determinations, the enthalpy change for the transition is independent of temperature, from 275 to 306 °K. The calculated values for the thermodynamic parameters determined and comparative values for relevant functional groups are shown in Table I. Our data suggest that the ionization of a tyrosyl hydroxyl group is intimately involved with this transition. The magnitudes of the parameters for the transition are considerably lower than those determined b y Barel and coworkers 5 for the thermal transition of lysozyme I/ill = + 1 2 o kcal/mole; xlS = + 3 4 7 cal. °C-l.mole -1 at 347 °K which presumably involves extensive disorganization of the tertiary structure of the molecule. Arguments have been presented in a preceeding communication 3 which implicate either tyrosine-2o or -23 as the site of the chromophore in the lysozyme derivative. Such a location is consistent with the spectroscopic changes that have been TABLE

I

THERMODYNAMIC PARAMETERS ASSOCIATED WITH THE STRUCTURAL TRANSITION IN OtYr-NDD LYSOZYME AND WITH THE IONIZATION OF RELEVANT FUNCTIONAL GROUPS

Compound Otyr-NBD-lys°zY Otyr-NBD-lys°zY Otyr-NBD-lys°zY 01~r-NBD-lys°zY Phenol 4-Hydr°xymethylp Lysine

me me me me henol

/1H kcal/mole

pKa

A S cal. °C -1.mole -1 Temp °K

Ref.

4.74 4.74 4.74 4.74 5.65 4.5 0.30 12.8o 11.6o

1°.13 9.99 9-89 9.76 9.79 9.84 2 . 1 8 (pK1) 8 . 9 5 (pK.,) lO.53 (pK,)

- - 2 9 . 9 4--29.9 ± - - 2 9 . 9 4- - 2 9 . 9 4-- 26.7 --30.o --9.0 2.0 --9.3 °

a a a a b b b b b

a, t h i s w o r k ; b, c o m p i l e d f r o m S o b e r 4.

0. 4 0.4 0. 4 0. 4

275-5 287.7 300.0 305. 7 298 298 298 298 298

34

A. ABODERIN et al.

observed in this study. Adjacent to this area is the large hydrophobie portion of the lysozyme molecule 6. In terms of these structural facts the conformational transition undergone by 0tyr-NBD-lysozyme can be summarized as one which is brought about as a result of the ionization of the hydroxyl group of another tyrosyl residue and which involves the transfer of the chromophore from an aqueous environment into a non-polar one. The group responsible for this conformational transition is probably not tyrosine residue 53, because of the relatively large distance of this residue from the chromophoric site in the tertiary structure and its separation from this site by the active site cleft. Alternative interpretations of the data are possible because of the tendency 7,8 of lysozyme to polymerize beyond the dimeric state at pH values higher than 9. In terms of this, the changes observed in this work could be due either to the sandwiching of the 0tyr-NBD ehromophore in the subunit contact surface from which water molecules have been expelled, or to a conformational change of the type already described, which, in this case, would be consequent upon polymerization. The fact, however, that the protein concentration at which the present experiments have been performed (o.o16%) is much lower than that ( > 2%) at which significant amounts of polymer can be expecte&, s renders these interpretations unlikely. Since 0t~r-NBD-lysozyme is enzymatically active and the overall conformation differs only slightly from that of the native enzyme s, it seems safe to conclude that the pH-dependent conformational transition observed with the modified protein is very similar to that which has been suggested for the native enzyme 1,2. The present results, therefore, suggest that both the change in the rotatory power at 199 nm and the changes in the quantum yield of the protein fluorescence emission might be better interpreted in terms of a conformational change which is a consequence of the ionization of one tyrosyl residue rather than being attributed solely to the ionization of a tyrosine residue itself 1. Also, it is likely that molecules in crystals of isoelectric lysozyme (obtained at high p H > 9.5) might show differences in Feptide back-bone conformation and in the orientation of certain side chains from those molecules in lysozyme hydrochloride crystals obtained at p H 4.7 which have been used for X-ray crystallographic studies 6. ACKNOWLEDGEMENT

We wish to thank Professor P. Pino for his interest and the Roche Research Foundation for generous support to A.A. during the course of this investigation. REFERENCES I Lehrer, S. S. a n d F a s m a n , G. D. (1967) J. Biol. Chem. 242 , 4644-4651 2 Halper, J. p,, L a t o v i t z k i , N., Bernstein, H. a n d Beychok, S. (1971) Proc. Natl. Acad. Sci. U.S. 68, 517-522 3 Aboderin, A. A., Boedefeld, E. a n d Luisi P. L. (1973) Biochim. Biophys. Acta 328, 20-30 4 Sober, H. A. (1968) Handbook of Biochemistry J 49-139 5 BareI, A. O., Prieels, J. P., Maes, E., Looze, Y. a n d Leonis, J. (1972) Biochim. Biophys Acta. 257, 288-296 6 Blake, C. C. F., Mair, G. A., N o r t h , A. C. T., Phillips, D. C. a n d Sarlna, Y. R. (I967) Proc. R. Soc. London, Ser. B, 167, 365-377 7 Sophianopoulous, A. J. a n d v a n Holde, K. E. (1964) J. Biol. Chem. 239, 2516-2524 8 t3ruzzesi, M., Chiancone, E. a n d A n t o n i n i , E. (1965) Biochemislrv 4, 1796 18oo 9 Fevold, H. L. a n d Alderton, G. (1949) Biochem. Prep. i, 67-71