J. Mol. Biol. (1976) 103, 799-801
LETTER
TO THE
EDITOR
Crystalline Bovine Liver Catalase A crystalline molecular
form of bovine liver 2.fold axes is incorporated
cat&se into
has been the crystal
found in which symmetry,
one
of the
Bovine liver catalase (EC 1.11.1.6) is a tetrameric enzyme which catalyzes the decomposition of hydrogen peroxide. It has a molecular weight of 250,000 (Sumner t Gralen, 1938) with one heme per subunit (Sund et al., 1967). Four different crystalline forms of the enzyme have been characterized by X-ray diffraction investigations. These have been summarized by McPherson & Rich (1973). In no case is the molecular symmetry incorporated into the crystal symmetry. However, electron microscopy and X-ray diffraction studies have led to the suggestion that the subunits are related by at least a single 2-fold axis (Glauser & Rossmann, 1966; Gurskaya et al., 1971) and probably by 222 symmetry (Vainshtein et al., 1968; Eventoff & Gurskaya, 1975). We report here the results of a preliminary X-ray diffraction study of a new crystal form of the enzyme, suitable for high-resolution structure analysis, which confirms the point group symmetry being at least 2. Catalase was purified from fresh beef liver by the method of Sumner BEDounce (1937)) and crystallized at room temperature from solutions which contained 0.05 M-Tris *HCl, 20% saturated sodium citrate and 5 mg catalase/ml adjusted to pH 8.5 to 9.0. The crystals, shaped in the form of parallelepipeds with roughly equal edges, could be grown to have body diagonals of up to O-4 mm. Their space group was found to be P3,21 or P3,21 with a = 142*3&0*6 A and c = 104.0*05 A. Assuming three molecules per unit cell, the volume-to-mass ratio (V,) is 2.42 A3/dalton. Furthermore, if catalase is assumed to be a sphere of approximately 80 A diameter, as indicated by electron microscopy (Valentine, 1964; Vainshtein & Kiselev, 1966; Labaw, 1967), the volume occupied by solvent: in this case, is 56%. Both of these results are well within the observed limits for other protein crystals (Matthews, 1968). Assuming hix molecules per unit cell leads to values for both the volume-to-mass ratio and solvent volume which are outside these normal ranges. The tetramer, therefore, must be positioned on a crystallographic 2-fold axis! in contrast to all previously found crystal forms where the whole molecule invariably formed the asymmetric unit. The three catalase molecules can be crudely represented as spheres of 80 A diameter placed at (x,0,1/3), (0,x,2/3), and (Z&O). Figure 1 represents the variation of the two shortest intermolecular contacts as a function of x within the limits 0 < x 5 0.5. The functions are reflected at 0.5. Any value of x can be considered acceptable if there is no intermolecular contact of less than 80 A. Hence Figure 1 shows that the permissable and unique range of x is 0.28 < x 5 0.44, within which there are no molecular contacts less than 80 A. A comparison of observed and calculated structure factors to a resolution of 30 A (Fig. 2) further conttrains the allowed range of x to be 0.38 5; x 5 0.42. A molecular envelope of 40 A radius gave best agreement with the 799
FIO. between molecular
1. The variation in ir~termulecular distance as a function the molecular centers at (z, 0, l/3) and (0, z, 2/X); c-1 centers at (zr. 0. l/3) anti (1 s. I J, 0).
of .r.
.j-d ~, t,he distance
, The tlistanco between t,he
32 30 28
&24 22 20 IS -’
16 14 12 IO 0 6 4
4x-------_
2 i 01
02 X
03 in froctlonol
04
05 co-ordinates
14’1~:. 2. Variation of the% amplitudr of the structure factors tion of I. The amplitudw were calculated assuming catalase Shown also are the obwrvrtl amplitudrs, on a suitably chosrn tlw calculat,ed valurs al .r ~ 0.39.
within a rcsolutiorl of 30 A tts a frmc. t,o bc a uniform sphere of 40 Ak radius. dative scale. which agree best with
observed data. The molecular packing arrangement which is thus produced differs from that observed in the orthorhombic P2,2,21 crystals (McPherson $ Rich, 1973; Unwin, 1975) or the related trigonal crystals found by Vainxhtein ef al. (1968). Longley (1967) and Rossmann & Labaw (1967). In the trigonal form, Eventoff &
LETTER
TO
THE
EI)ITOL1
Ml I
Gurskaya (197.5) have shown tha,t the three molecular Sfold axes are inclined roughly equally to the crystallographic 3, axis. In the present crystal form, however, one of the molecular dyads is perpendicular to the crystallographic 31 axis. Furthermore, t,he crystallographic axes int’errelate the different molecules in the orthorhombic and the earlier trigonal crystals, whereas. in the present’ crystals the molecules sit on the crystallographic S-fold axes. We are now collecCng three-dimensional data from the native crystals, using oscillation photograph,v. for a high-resolution structure determination. \Vt> tlwlk supported Yriww
Chris by tile Foundation
Kraemer National (grant
for help in the preparation Institutes of Health (grant no BMS74-23537).
Department of Biological Sciences, M’cst Lafayette. Ind. 47907. U.S.A. I&~wi~c~tl
18 Drccmbrr
Purdue
IJniversitl
of tile manuscript. The work was no. GM 10704) and the Nat,ional
\VILLIAM
EVENTOFF
NosuoTAi-xK~ MImAm G.
R~S~MANN
197::
REFERENCES EvwtotY, Glauser, (:urskaya,
W. $ Gurskaya,, G. V. (1975). J. Mol. Biol. S. & Rossmann, M. G. (1966). Acta Crystallogr. G. V., Lobanova, (:. M. & Vainshtein.
93. 55. 62. 21, 175-176. B. K. (1971).
Kristullografiya.
Labaw. L. W. (1967). J. Ultrastruct. Bes. 17, 327-341. Longlel\, W. (1967). ,J. Mol. Bid. 30, 323-327. Matthrws, B. W. (1968). J. Mol. Biol. 33, 491. 497. McPllrrson, A. Jr & Rich, A. (1973). Arch. Biochem. Biophys. 157, 23-27. Rossmann. M. (1. & Labaw, L. W. (1967). J. Mol. BioZ. 29, 315-316. Surnt~w, ,J. B. & Dounce, A. L. (1937). J. Biol. Chem. 121. 417-424. Sumller, .I. B. & GralBn. M. (1938). J. Biol. Chem. 125, 33-36. Sutrd. H.. Webcsr, K. & Melbert. E. (1967). Eur. J. Biochem. 1, 400 410. Unwin, I’. N. T. (1975). J. 11lol. Biol. 98, 235-242. Vainslltein, B. K. & Kiselev, N. A. (1966). In Proceedings of the 6th Internation,a/ ,for Electron Microscopy, p. 17, Maruzen Co. Ltd.. Nihonbashi. Tokyo. \‘ainshtjrin, B. K., Barynin. V. V. & Gurskaya. G. V. (1968). IIoxll. Akad. Satck 182 ,1 569 . I57” _, \.illvtktiw. K. (‘. ( 1!)64). S&we ( Lovhn). 204. 1262 1264.
16.
Congress S.S.S.K.
LETTER
TO THE
EDITOR
Crystalline Bovine Liver Catalase A crystalline molecular
form of bovine liver 2.fold axes is incorporated
cat&se into
has been the crystal
found in which symmetry,
one
of the
Bovine liver catalase (EC 1.11.1.6) is a tetrameric enzyme which catalyzes the decomposition of hydrogen peroxide. It has a molecular weight of 250,000 (Sumner t Gralen, 1938) with one heme per subunit (Sund et al., 1967). Four different crystalline forms of the enzyme have been characterized by X-ray diffraction investigations. These have been summarized by McPherson & Rich (1973). In no case is the molecular symmetry incorporated into the crystal symmetry. However, electron microscopy and X-ray diffraction studies have led to the suggestion that the subunits are related by at least a single 2-fold axis (Glauser & Rossmann, 1966; Gurskaya et al., 1971) and probably by 222 symmetry (Vainshtein et al., 1968; Eventoff & Gurskaya, 1975). We report here the results of a preliminary X-ray diffraction study of a new crystal form of the enzyme, suitable for high-resolution structure analysis, which confirms the point group symmetry being at least 2. Catalase was purified from fresh beef liver by the method of Sumner BEDounce (1937)) and crystallized at room temperature from solutions which contained 0.05 M-Tris *HCl, 20% saturated sodium citrate and 5 mg catalase/ml adjusted to pH 8.5 to 9.0. The crystals, shaped in the form of parallelepipeds with roughly equal edges, could be grown to have body diagonals of up to O-4 mm. Their space group was found to be P3,21 or P3,21 with a = 142*3&0*6 A and c = 104.0*05 A. Assuming three molecules per unit cell, the volume-to-mass ratio (V,) is 2.42 A3/dalton. Furthermore, if catalase is assumed to be a sphere of approximately 80 A diameter, as indicated by electron microscopy (Valentine, 1964; Vainshtein & Kiselev, 1966; Labaw, 1967), the volume occupied by solvent: in this case, is 56%. Both of these results are well within the observed limits for other protein crystals (Matthews, 1968). Assuming hix molecules per unit cell leads to values for both the volume-to-mass ratio and solvent volume which are outside these normal ranges. The tetramer, therefore, must be positioned on a crystallographic 2-fold axis! in contrast to all previously found crystal forms where the whole molecule invariably formed the asymmetric unit. The three catalase molecules can be crudely represented as spheres of 80 A diameter placed at (x,0,1/3), (0,x,2/3), and (Z&O). Figure 1 represents the variation of the two shortest intermolecular contacts as a function of x within the limits 0 < x 5 0.5. The functions are reflected at 0.5. Any value of x can be considered acceptable if there is no intermolecular contact of less than 80 A. Hence Figure 1 shows that the permissable and unique range of x is 0.28 < x 5 0.44, within which there are no molecular contacts less than 80 A. A comparison of observed and calculated structure factors to a resolution of 30 A (Fig. 2) further conttrains the allowed range of x to be 0.38 5; x 5 0.42. A molecular envelope of 40 A radius gave best agreement with the 799
FIO. between molecular
1. The variation in ir~termulecular distance as a function the molecular centers at (z, 0, l/3) and (0, z, 2/X); c-1 centers at (zr. 0. l/3) anti (1 s. I J, 0).
of .r.
.j-d ~, t,he distance
, The tlistanco between t,he
32 30 28
&24 22 20 IS -’
16 14 12 IO 0 6 4
4x-------_
2 i 01
02 X
03 in froctlonol
04
05 co-ordinates
14’1~:. 2. Variation of the% amplitudr of the structure factors tion of I. The amplitudw were calculated assuming catalase Shown also are the obwrvrtl amplitudrs, on a suitably chosrn tlw calculat,ed valurs al .r ~ 0.39.
within a rcsolutiorl of 30 A tts a frmc. t,o bc a uniform sphere of 40 Ak radius. dative scale. which agree best with
observed data. The molecular packing arrangement which is thus produced differs from that observed in the orthorhombic P2,2,21 crystals (McPherson $ Rich, 1973; Unwin, 1975) or the related trigonal crystals found by Vainxhtein ef al. (1968). Longley (1967) and Rossmann & Labaw (1967). In the trigonal form, Eventoff &
LETTER
TO
THE
EI)ITOL1
Ml I
Gurskaya (197.5) have shown tha,t the three molecular Sfold axes are inclined roughly equally to the crystallographic 3, axis. In the present crystal form, however, one of the molecular dyads is perpendicular to the crystallographic 31 axis. Furthermore, t,he crystallographic axes int’errelate the different molecules in the orthorhombic and the earlier trigonal crystals, whereas. in the present’ crystals the molecules sit on the crystallographic S-fold axes. We are now collecCng three-dimensional data from the native crystals, using oscillation photograph,v. for a high-resolution structure determination. \Vt> tlwlk supported Yriww
Chris by tile Foundation
Kraemer National (grant
for help in the preparation Institutes of Health (grant no BMS74-23537).
Department of Biological Sciences, M’cst Lafayette. Ind. 47907. U.S.A. I&~wi~c~tl
18 Drccmbrr
Purdue
IJniversitl
of tile manuscript. The work was no. GM 10704) and the Nat,ional
\VILLIAM
EVENTOFF
NosuoTAi-xK~ MImAm G.
R~S~MANN
197::
REFERENCES EvwtotY, Glauser, (:urskaya,
W. $ Gurskaya,, G. V. (1975). J. Mol. Biol. S. & Rossmann, M. G. (1966). Acta Crystallogr. G. V., Lobanova, (:. M. & Vainshtein.
93. 55. 62. 21, 175-176. B. K. (1971).
Kristullografiya.
Labaw. L. W. (1967). J. Ultrastruct. Bes. 17, 327-341. Longlel\, W. (1967). ,J. Mol. Bid. 30, 323-327. Matthrws, B. W. (1968). J. Mol. Biol. 33, 491. 497. McPllrrson, A. Jr & Rich, A. (1973). Arch. Biochem. Biophys. 157, 23-27. Rossmann. M. (1. & Labaw, L. W. (1967). J. Mol. BioZ. 29, 315-316. Surnt~w, ,J. B. & Dounce, A. L. (1937). J. Biol. Chem. 121. 417-424. Sumller, .I. B. & GralBn. M. (1938). J. Biol. Chem. 125, 33-36. Sutrd. H.. Webcsr, K. & Melbert. E. (1967). Eur. J. Biochem. 1, 400 410. Unwin, I’. N. T. (1975). J. 11lol. Biol. 98, 235-242. Vainslltein, B. K. & Kiselev, N. A. (1966). In Proceedings of the 6th Internation,a/ ,for Electron Microscopy, p. 17, Maruzen Co. Ltd.. Nihonbashi. Tokyo. \‘ainshtjrin, B. K., Barynin. V. V. & Gurskaya. G. V. (1968). IIoxll. Akad. Satck 182 ,1 569 . I57” _, \.illvtktiw. K. (‘. ( 1!)64). S&we ( Lovhn). 204. 1262 1264.
16.
Congress S.S.S.K.
