Structure of horse liver alcohol dehydrogenase

ARCHIVES

OF BIOCHEMISTRY

Structure

AND

of Horse

I. Structural

112, 215-217 (19%)

BIOPHYSICS

Liver Alcohol

Symmetry

and Conformational

CARL-IVAR Depahnent

of Agricultural

Dehydrogenase

BRANDfiN

Chemistry I, Agricultural Received

Changes

College of Sweden, Uppsala,

Sweden

June 30, 1965

The paper describes a preliminary X-ray investigation of crystals of horse-liver alcohol dehydrogenase and various binary and ternary complexes formed between this enzyme, its coenzyme, and some inhibitors. Molecules of free enzyme and its complex with coenzyme contain two identical subunits, each of molecular weight approximat,ely 42.000, which are related by a twofold symmetry axis. Inhibitor molecules like pyrazole and isobutyramide induce conformational changes in the enzyme molecule.

Horse-liver alcohol dehydrogenase is a zinc metalloenzyme of molecular weight 84.000 (1). Each enzyme molecule requires two molecules of NAD to attain full activity (2). Extensive physicochemical studies have shown t.hat, binary and ternary complexes of enzyme, coenzyme, and various inhibitor molecules may be obtained in crystalline form (3, 4). The aim of the present investigation was to explore the possibilities of obtaining information about the physical stat.e of t.his enzyme by t.he use of X-ray crystallography without making a complete structure analysis. A preliminary report of some of the results has been published (5). MATERIALS

AND

the binary complex of enzyme and coenzyme both show orthorhombic symmetry and, within the limits of error, identical cell dimensions. Since the observed space group, C2221, has eight nonequivalent positions, a brief ealeulation shows that. each asymmetTABLE CRYSTAL

ADH

B = ER?

Orthorhombic symmetry Space group C2221 a=56A b=75;i c = 181 B

METHODS V = 760.000 it3 z=a d = 1.2 gm/cm3

All the crystals used in this investigation were provided by Theorell and his co-workers. The preparation and crystallization procedures have been described earlier (4). The conditions of crystallization were identical for all the crystals investigated. The crystals obtained in this way were fragile and unsuitable for accurate X-ray work since they were somewhat disordered. However, they were adequate for a preliminary investigation of the cell-dimensions and symmetry of these Copper radiation was used to take complexes. nine-degree precession photographs.

I

I)ATA~ OF HORSE LIVER AND SOME COMPLEXES E(RZ)z = E(OP)> = E(OPI)z

Monoclinic symmetry Space group P2, a = 51 A b=44i% c = 182i y = 108” V = 388.000 ;i3 z=2 d = 1.2 gm/cm3

alcohol dehydrogenase; GE = Horse liver 0 = NAD+ = nicotinamide adenine dinucleotide; R = NADH; I = isobutyramide; P = pyrazole; PJ = 3-iodopyrazole.

unit has a weight of 68.700 on the atomic mass scale; t,his is less than the molecular weight, 84.000, of the enzyme. Each molecule must therefore be located on the dyad axes of the space group, and consequently each asymmetric unit contains only one half of the molecule. It may thus be safely ric

RESULTS

Table I summarizes the results obtained in this investiga.tion. The free enzyme and 215

216

FIG. la

Monoclinic axes FIG. lb FIG. 1. (a) A projection of the centered orthorhombic unit cell of free ADH and the corresponding primitive monoclinic unit cell (dotted lines). (b) A projection of the primitive monoclinic unit cell of the ternary complexes. (Shadowed objects are related to the white ones by a translation along c of half the length of the c-axis.) Orthorhombic

axes

concluded that the molecule of horse-liver alcohol dehydrogenase contains two subOunits crystallographically identical at a 6-A resolution. This view is in agreement with recent chemical work on carboxymethylated alcohol dehydrogenase which has shown that in all probability this enzyme consists of two identical polypeptide chains (6, 7). In contrast t’o the above, the ternary complexes which have been investigated show monoclinic symmetry. Each asymmetric unit thus has a weight of 140.000 and cont’ains one enzyme molecule of molecular

weight approximately 85.000. The only element of symmetry in this st,ructure is a twofold screw-axis which relates two such molecules in the unit cell. These two types of unit cell have rather different cell dimensions; nevertheless, they show a very close relationship. The c-axes are approximately of the same length in both unit cells. Projections of the orthorhombic and monoclinic unit cells along the c-axes are shown in Figs. la and lb, respectively, with the space-group symmet,ry operating on an object with two subunits.

HORSE

LIVER

ALCOHOL

If the t,wofold symmetry axes along a and b in the cent.ered orthorhombic unit. cell are removed, a primitive monoclinic unit cell, with the space-group symmetry 1’21, is obtained (Fig. la). The al- a;d &-axes of this cell are both qua1 to 47 A, and the angle y between thcsc axes is 106.5”. Since the cell dimensions of this hypothetical monoclinic unit-cell are very similar to t.hosc actually obtained for the ternary con~plexes, t.herc seems little doubt, t,hat close similarities must exist betwocn the molecular arrangemcnts of the two types of unit cells investigated. The main difference observed is that, the crystallographic symmetry which relates the two subunits in the free enzyme and in t,he binary complex is absent from the ternary complex. The symmet.ry change induced by the inhibit.or molecules may be caused either by slightly displacing t,he whole molecule from the twofold axis in t.he crystal lattice while preserving t.he molecular shape, or by a conformational change of the molecule. The first alternative would correspond to a pure packing effect caused by the inhibitors in which t.he main arrangement of the molecules is preserved but with slightly different intermolecular contacts. It. seems improbable that the small inhibitor molecules arc involved in the packing of the complexes since the much larger coenzyme molecules do not. cause any change in the packing. 13oth the inhibitor and the coenzyme are presumably bonded rather closely to each other at the active site. It ~ecms much more probable that the inhibitor molecules induce a conformational c*hangc of the wzymc molecules, especially in view of an optical rotary dispersion study of ADH and its complexes in solut,ion (8). A large difference has been recorded between the dispersion curves of the ternary complexes on t hc one hand and t.hosc of the enzyme and its binary complexes on the other. This conformational change may involve either a different orientation of the subunits relative to each other or a change in the configuration of the peptide chains or a combination of t.hese LWOeffects.

217

I)ElIYJ)I~OGENA8P:

Conformational changes in protein molccules probably play an important role in their mode of action. Muirhead and Perutz (9) have shown t.hat, the subunits of hcmoglobin exhibit differences of relative orientation in the rcduwd and oxy forms. Monad et al. (10) have recently suggested that. conformational changes arc csscntial in the rcgulation of metabolic activit.y, and Tioshland (11) has discussed and studied r;uc+ changes at t,hc a&w sit.c during enzyme action. Further crystallographic~ work aimed at. a mow detailed study of these conformational changes in alcohol dchydrogenasc is now in progress.

I wish to thank Professor I. Lindqvist. for all the facilities placed at my disposal and for his stimulating interest in this work. Sincere thanks arc also due to Professor IIugo Theorell, 1)r. T. Yonet.ani, and Dr. :I. ;lkcson at the Nobel Medical Institute in Stockholm for providing the crystals, and to Mr. L.-M. Larason for help with the S-ray work. This investigation was supported by Grant Y128b from the Swedish Medical Research Council and by 1J.S. Public IIealth Service research grant (;M 11307 from the Dcpartmcnt of Health, Bducation, and Wclfarc. REFERESCP:S 1.

~‘:HHEXBEKG,

Stand.

2.

A.,

AND

~)ALZIEL,

K.,ncta Chen.

12, 365 (1958).

JJ., ANV THF.OIIEI.I., II., in “The Enzymes” (I’. Boycr, H. Lardy, and K. Myrbgck, eds.), 2nd edition, Vol. 7, p. 25. Academic Press, New York (1962). II., AND YosE,r.ih.l, T., Binioche~l. 3. THEOHELL, z. 338, 537 (1963). T., .~NI) THEORELL, H., :1fch. 4. Yoh.ETANI, Niochem. Biophys. 100, 554 (19G3). C.-I., IAISSON, L.%., LISDQVIST, 5. %thNDEN, I., TIIEOILELI., II., .4s1) YOSETASI, ‘I’., Arch. Wiochem. Wiophys. 103, 195 (1965). ANV VALLEE, B. I,., Hiochemistry G. LI, T.-K., 3, 869 (1964). I., .\‘alure 203, 30 (l!Xi-l). 7. H.\RHIS, A., THEONELL, IT., .\xv YONE8. ROSENBERG, TANI, T., :\‘UfILre 203, 755 (19&t). H., ASI) PERUTZ, M. F., .\-atltre 9. ~IIXILIIEAD, 193, c,33 (1963). J., CIIANGEUX, J.-P., ASI) .JACOB, I”., 10. ~IOSOD, J. Mol. Rio/. 6, 306 (1993). L). E., JK., F’ederalion Proc. 23, 11. KOSIIIAND, 719 (1964). SUSD,