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Preliminary crystallographic studies on quinolinate phosphoribosyltransferase
J. Mol. Biol. (1977) 117, 1101-1107
Preliminary Crystallographic Studies on Quinolinate Phosphoribosyltransferase Quinolinate phosphoribosyltransferase isolated from pig liver was crystallized in two different forms. Type I crystals, grown at 4°C, had a trigonat lattice of dimensions a = b = 178 A, c = 132 ~ but diffracted poorly. Type I I crystals grew at room temperature and diffracted to about 2.2 ~_ resolution. The space group was determined as P6322 with a = b = 120-9 A, c = 95-0 ~. The space group symmetry shows the molecules to be hexamerie with 32 point symmetry, in contrast to the earlier pentamerie arrangement based on molecular weight determinations. The de novo synthesis of nicotinic acid mononucleotide from quinolinic acid and phosphoribosyl pyrophosphate is common to plant, bacterial and mammalian systems (Ortega & Brown, 1960; Gholson, 1966; Ahmad & Moat, 1966; Isquith & Moat, 1966; Ogasawara et al., 1967; Scott et al., 1969). This reaction is catalyzed by the enzyme quinolinate phosphoribosyltransferase (Mr, 172,000; EC 2.4.2.19) and involves the formation of a quaternary ring E-N-glycoside, a pyrophosphorolytie cleavage and an aromatic ring deearboxylation (Fig. 1). Since nicotinic acid mononucleotide is an obligatory intermediate in the synthesis of the pyridine nueleotide group of coenzymes, quinolinate phosphoribosyltransferase functions centrally in this important pathway.
~ '~" N ~ /C02a-PP-ribose-P + ~/'~"] ' ~
~/C02-
Mgz+ >-
COz+ PPi
COz-
I
-
Ouinotinic acid
ribose-P
Nicotinic
acid mononucleofide
FIG. 1. The reaction catalyzed by quino]inate phosphoribosyltransfcrase. The biosynthesis of quinolinie acid in mammals occurs aerobically via tryptophan degradation. However, recent evidence has suggested that vestiges of the anaerobic system, characteristic of more primitive organisms, still exist in mammalian tissues (Sakakibara et al., 1977). Furthermore, the anaerobic pathway of pyridine coenzyme synthesis has been postulated to be one of the earliest developments in biochemical evolution (Gaertner & Shetty, 1977). Since both aerobic and anaerobic routes converge at the level of quinolinic acid, quinolinate phosphoribosyltransferase must have developed as part of the anaerobic pathway. Consequently, quino]inate phosphoribosyltransferase embodies structural information developed during the earliest stages of evolution of the NAD biosynthetic pathway. 1101
1102
W. DONALD L. MUSICK
This preliminary report describes the crystallization, space group identification and molecular symmetry determination of quinolinate phosphoribosyltransferase isolated from pig liver. Pig livers (7 kg) were frozen immediately after slaughter and stored at --20°C. The enzyme was isolated essentially by the method of Taguchi & Iwai (1975a) yielding approximately 200 mg of microcrystalline protein. Enzyme activity was measured speetrophotometrically using the assay conditions of Taguchi & Iwai (1975b) and a modification of the cyanide adduet technique of Packman & Jakoby (1967). Under these conditions, the specific activity of the microerystalline enzyme was identical to that reported using the quinolinate radioassay (Taguehi & Iwai, 1975a). A preliminary search for conditions favoring the growth of large single crystals was carried out in mierodialysis buttons. A suspension of twice reerystallized quinolinate phosphoribosyltransferase was pelleted by centrifugation and the sedimented crystals dissolved in potassium phosphate buffer (50 m.u, pH 7"0) to concentrations of 24 lng/ml and 8 mg/ml. The concentrations of ammonium sulfate used as precipitant were initially selected as 10% to 30% saturation. The range of p H chosen in this survey was based on the isoeleetrie point of the enzyme (4-3), the pH optimum (6.1) and the pH at which mierocrystals are obtained (7.0) (Taguehi & Iwai, 1975b). Citrate-phosphate buffer was chosen ( ~ 5 0 mM) to cover this range and the crystallization survey was carried out at 4°C. In several days, large (~-~0.5 ram) glassy hexagonal plates and spurs of fine needles grew in buttons containing 10% ammonium sulfate at p H 7.0 at either protein concentration. All other conditions precipitated the protein. Subsequent attempts at growing single large quinolinate phosphoribosyltransferase crystals were carried out in phosphate or Tris buffer over a pH range of 6.7 to 9-0 using ammonium sulfate or sodium citrate. Crystals grew at all pH values tested; however, the concentration of salt required for crystallization increased with increasing pit. The crystals grew as platy aggregates with a glassy appearance at p i t values below 7.5 (protein concentration approx. 20 mg/ml). Above pH 7.5, the crystals appeared as large hexagonal plates growing in fan-shaped arrays. Each plate grew singly to dimensions greater than 1.0 mm across and approximately 0.2 mm thick (Fig. 2). Since single plates could be isolated, trial diffraction photographs were taken perpendicular to the hexagonal face (h/c0). The fragile plates were mounted in thin-walled glass capillaries and placed in a Buerger precession camera with an Elliott GX-6 rotating anode X-ray source. A two-hour exposure with /x = 2 ° is shown in Figure 3. Cell constants determined from this photograph and the hO1 zone identified a trigonal lattice with a = b -- 178 A, c = 132 A. Unfortunately, the diffraction pattern did not extend beyond 40 A resolution for crystals growl from either ammonium sulfate or sodium citrate. A second crystal form (type II) was grown at room temperature over a pH range of 7"5 to 8-5. Mierodialysis cells containing quinolinate phosphoribosyltransferase at approximately 20 mg/ml were dialyzed against 50 mM-Tris buffer with sodium citrate at an ionic strength of 1.2 to 1.6. Large hexagonal rods grew in the cells in several hours (Fig. 4). The same crystal form was also obtained using the technique of vapor diffusion with ammonium sulfate as precipitant. Again, large hexagonal rods grew in several hours. The crystallization conditions were refined using mierodialysis buttons to an optimal ionic strength of 0.8 in citrate at pH 8.0 in 50 m~-Tris, t{C1 buffer and a protein
L E T T E R S TO THE E D I T O R
ll03
Fic. 2. Type I quinolinate phosphoribosyltransferase crystals. Grown at 4°C in 50 raM-potassium phosphate buffer (pl-I 7.0), 14~o saturation ammonium sulfate and 23 mg quinolinate phosphoribosyltransferase/ml. concentration of 20 to 25 mg/ml. Under these conditions, the enzyme was completely soluble at 4°C but produced large hexagonal rods ( > 2 m m long) at room temperature in one to two weeks. I n addition to the hexagonal apoenzyme crystals, a pseudo-ternary complex with the identical morphology has been grown. This complex contains Mg 2+-phosphoribosyl pyrophosphate and the competitive inhibitor phthalic acid (Taguchi & Iwai, 1976). I f the molecule undergoes a conformational change (Paekman & Jakoby, 1965,1967; Mann & Byerrum, 1974) on binding this complex, the change does not alter the crystal morphology. The ape crystals were found to diffract to as far as 2"2 A resolution with sealed tube Cu radiation. The space group of this crystal form was subsequently identified as P6322 with a --~ b = 120.9 A, c -- 95-0 A. High resolution precession photographs (t* = 16°) of the hkO and hOl zones are shown in Figure 5. Assuming a molecular weight of 172,000, Vm values of 3.59, 2-39 and 1.79 A3/dalton are calculated for two, three and four molecules per unit cell, respectively. All of these ratios are within the acceptable range for protein crystals (Matthews, 1968). Since the space group has 12 equivalent positions, the unit cell will accommodate only two or four molecules in special positions. A consequence of locating the molecule in these positions is that it is necessarily hexameric. This is in contradiction to the five subunit model proposed by Taguehi & Iwai (1975a). Furthermore, since several different special positions are possible, the molecule m a y belong only to point s y m m e t r y group 3, 32 or 6. A two-molecule unit cell makes the crystallographic asymmetric unit one-
1101
W. D O N A L D L, M U S I C K
FIG. 3. /s = 2° precession photograph of type I quinolinate phosphoribosyltransferase taken perpendicular to the hexagonal face (hkO): a 2 h exposure on a 75 mm Buerger preecssion camera on an Elliott GX-6 rotating anode X-ray source (35 kV, 18 mA, Ni-filtered CuKa radiation). sixth of the quinolinate phosphoribosyltransferase molecule, whereas one-third of the molecule is the asymmetric unit in a four-molecule celt. However, the latter ease requires t h a t two molecules be placed along the z axis, allowing a maximal molecular dimension of 47 A in this direction. I f the quinolinate phosphoribosyltransferase molecule is approximated as a sphere of uniform density with a partial specific volume of 0.74 ml/g (Taguehi & Iwai, 1975a), a molecular diameter of 71 A is calculated. Since equilibrium eentrifugation studies have indicated t h a t the enzyme is a compact protein (Packman & J a k o b y , 1967; Taguchi & Iwai, 1975a), this approximation is justifiable. Therefore, a four-moleeule unit cell is an unlikely possibility. Furthermore, one of the two special positions allowed in a two-molecule unit cell also stacks two molecules on the z axis. Consequently, only the enantiomorphie positions (1/3, 2/3, 1/4; 2/3, 1/3, 3/4) or (2/3, 1/3, 1/4; 1/3, 2/3, 3/4) are reasonable locations for the molecule. These special positions require a molecular point s y m m e t r y of 32. An a t t e m p t to put these arguments on a more quantitative basis was made b y calculating intensities based on a model structure and comparing them to the observed low-order reflections. The model was a constant density sphere of variable radius
LETTERS
TO T H E E D I T O R
1105
FIG. 4. Type I I quinolinate phosphoribosyltransferase crystals. Grown at room temperature in 50 mN-Tris buffer (pit 8"0) at an ionic strength of 1.2 in citrate and 20 mg quinolinate phosphoribosyltransferase]ml.
placed in the special positions dictated by the two and four-molecule unit cells. The model which best fit the data contained two spheres of approximately 70 _£_diameter at centers 1/3, 2/3, 1/4 and 2/3, 1/3, 3/4. All models placing two molecules on the z axis poorly predicted the low-order intensities. A further refinement of the model was performed using a cluster of spheres with 32 symmetry at the indicated centers. The only constraint utilized was that the top trimerie aggregate of spheres be in contact with the bottom aggregate. The sphere radii were allowed to vary along with the radial separation of each trimerie aggregate. This model produced a better fit to the data with an optimal sphere radius of 19 A and a radial separation of 32 A. A diagram of this packing arrangement is shown in Figure 6. As seen in Figure 4, the hexad axis runs along the length of the crystal. The Laue symmetry of the lattice reduces the unique portion of reciprocal space to a 30 ° wedge along this axis. Consequently, this crystal form of quinolinate phosphoribosyltransferase is particularly well suited to the technique of oscillation photography and facilitates rapid data collection, which is now underway. A study of the amino acid sequence of quinolinate phosphoribosyltransferase has been started at Purdue University (3/[. Hermodson, personal communication) and thus far cyanogen bromide fragments as well as lysine and arginine-blocked tryptic peptides have been characterized. Preliminary sequencing studies of these fragments are now- in progress. 72
©
cD
®
~o
v
~
~,.~
~N ~
o
0 . ~
~
N
~.~
N
~
L E T T E R S TO T H E E D I T O R
1107 o
¼ FIG. 6. The packing arrangement of the quinolinate phosphoribosyltransferase, 32 hexamers deduced from low order reflections. I am grateful to Drs M. G. Rossmann, A. Sieignano and J. E. Johnson for helpful suggestions and comments, and to Sharon S. Wilder for help in the preparation of the manuscript. The work was supported by the National Institutes of Health (grant no. GM 10704) and the National Science Foundation (grant no. BMS74-23537). Departmertt of BiologieM Sciences Purdue University West Lafayette, Ind. 47907, U.S.A.
W . DONALD L. MUSlCK
]~eceived 3 August 1977 REFERENCES Ahmad, F. & Moat, A. G. (1966). J. Biol. Chem. 241,775 780. Gaertner, F. H. & Sherry, A. S. (1977). Biochim. Biophys. Aeta, 482, 453-460. Gholson, R. K. (1966). Nature (London), 212, 933-934. Isquith, A. J. & Moat, A. G. (1966). Biochem. Biophys. Res. Commun. 22, 565-571. Mann, D. F. & Byerrum, 1~. U. (1974). J. Biol. Chem. 249, 6817-6823. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Ogasawara, N., Chandler, J. L. 1~., Gholson, R. K., t~osser, R. J. & Andreoli, A. J. (1967). B~ochim. Biophys. Aeta, 141, 199-201. Ortega, ~ . V. & Brown, G. M. (1960). J. Biol. Chem. 235, 2939-2945. Packman, P. M. & Jakoby, W. B. (1965). Biochem. Biophys. Res. Commun. 18, 710-715. Packman, P. M. & Jakoby, W. B. (1967). J. Biol. Chem. 242, 2075-2079. Sakakibara, S., Wicks, F. D. & Gholson, 1~. K. (1977). Biochem. Biophys. Res. Commun. 76, 158-166. Scott, T. A., Bellion, E. & Mattey, M. (1969). Eur. J. Biochem. 10, 318-323. Taguchi, I~I. & Iwai, K. (1975a). Agr. BioL Chem. 39, 1493-1500. Taguehi, I-I. & Iwai, K. (1975b). Agr. Biol. Chem. 39, 1599-1604. Taguehi, H. & Iwai, K. (1976). Agr. Biol. Chem. 40, 385-389.
Preliminary Crystallographic Studies on Quinolinate Phosphoribosyltransferase Quinolinate phosphoribosyltransferase isolated from pig liver was crystallized in two different forms. Type I crystals, grown at 4°C, had a trigonat lattice of dimensions a = b = 178 A, c = 132 ~ but diffracted poorly. Type I I crystals grew at room temperature and diffracted to about 2.2 ~_ resolution. The space group was determined as P6322 with a = b = 120-9 A, c = 95-0 ~. The space group symmetry shows the molecules to be hexamerie with 32 point symmetry, in contrast to the earlier pentamerie arrangement based on molecular weight determinations. The de novo synthesis of nicotinic acid mononucleotide from quinolinic acid and phosphoribosyl pyrophosphate is common to plant, bacterial and mammalian systems (Ortega & Brown, 1960; Gholson, 1966; Ahmad & Moat, 1966; Isquith & Moat, 1966; Ogasawara et al., 1967; Scott et al., 1969). This reaction is catalyzed by the enzyme quinolinate phosphoribosyltransferase (Mr, 172,000; EC 2.4.2.19) and involves the formation of a quaternary ring E-N-glycoside, a pyrophosphorolytie cleavage and an aromatic ring deearboxylation (Fig. 1). Since nicotinic acid mononucleotide is an obligatory intermediate in the synthesis of the pyridine nueleotide group of coenzymes, quinolinate phosphoribosyltransferase functions centrally in this important pathway.
~ '~" N ~ /C02a-PP-ribose-P + ~/'~"] ' ~
~/C02-
Mgz+ >-
COz+ PPi
COz-
I
-
Ouinotinic acid
ribose-P
Nicotinic
acid mononucleofide
FIG. 1. The reaction catalyzed by quino]inate phosphoribosyltransfcrase. The biosynthesis of quinolinie acid in mammals occurs aerobically via tryptophan degradation. However, recent evidence has suggested that vestiges of the anaerobic system, characteristic of more primitive organisms, still exist in mammalian tissues (Sakakibara et al., 1977). Furthermore, the anaerobic pathway of pyridine coenzyme synthesis has been postulated to be one of the earliest developments in biochemical evolution (Gaertner & Shetty, 1977). Since both aerobic and anaerobic routes converge at the level of quinolinic acid, quinolinate phosphoribosyltransferase must have developed as part of the anaerobic pathway. Consequently, quino]inate phosphoribosyltransferase embodies structural information developed during the earliest stages of evolution of the NAD biosynthetic pathway. 1101
1102
W. DONALD L. MUSICK
This preliminary report describes the crystallization, space group identification and molecular symmetry determination of quinolinate phosphoribosyltransferase isolated from pig liver. Pig livers (7 kg) were frozen immediately after slaughter and stored at --20°C. The enzyme was isolated essentially by the method of Taguchi & Iwai (1975a) yielding approximately 200 mg of microcrystalline protein. Enzyme activity was measured speetrophotometrically using the assay conditions of Taguchi & Iwai (1975b) and a modification of the cyanide adduet technique of Packman & Jakoby (1967). Under these conditions, the specific activity of the microerystalline enzyme was identical to that reported using the quinolinate radioassay (Taguehi & Iwai, 1975a). A preliminary search for conditions favoring the growth of large single crystals was carried out in mierodialysis buttons. A suspension of twice reerystallized quinolinate phosphoribosyltransferase was pelleted by centrifugation and the sedimented crystals dissolved in potassium phosphate buffer (50 m.u, pH 7"0) to concentrations of 24 lng/ml and 8 mg/ml. The concentrations of ammonium sulfate used as precipitant were initially selected as 10% to 30% saturation. The range of p H chosen in this survey was based on the isoeleetrie point of the enzyme (4-3), the pH optimum (6.1) and the pH at which mierocrystals are obtained (7.0) (Taguehi & Iwai, 1975b). Citrate-phosphate buffer was chosen ( ~ 5 0 mM) to cover this range and the crystallization survey was carried out at 4°C. In several days, large (~-~0.5 ram) glassy hexagonal plates and spurs of fine needles grew in buttons containing 10% ammonium sulfate at p H 7.0 at either protein concentration. All other conditions precipitated the protein. Subsequent attempts at growing single large quinolinate phosphoribosyltransferase crystals were carried out in phosphate or Tris buffer over a pH range of 6.7 to 9-0 using ammonium sulfate or sodium citrate. Crystals grew at all pH values tested; however, the concentration of salt required for crystallization increased with increasing pit. The crystals grew as platy aggregates with a glassy appearance at p i t values below 7.5 (protein concentration approx. 20 mg/ml). Above pH 7.5, the crystals appeared as large hexagonal plates growing in fan-shaped arrays. Each plate grew singly to dimensions greater than 1.0 mm across and approximately 0.2 mm thick (Fig. 2). Since single plates could be isolated, trial diffraction photographs were taken perpendicular to the hexagonal face (h/c0). The fragile plates were mounted in thin-walled glass capillaries and placed in a Buerger precession camera with an Elliott GX-6 rotating anode X-ray source. A two-hour exposure with /x = 2 ° is shown in Figure 3. Cell constants determined from this photograph and the hO1 zone identified a trigonal lattice with a = b -- 178 A, c = 132 A. Unfortunately, the diffraction pattern did not extend beyond 40 A resolution for crystals growl from either ammonium sulfate or sodium citrate. A second crystal form (type II) was grown at room temperature over a pH range of 7"5 to 8-5. Mierodialysis cells containing quinolinate phosphoribosyltransferase at approximately 20 mg/ml were dialyzed against 50 mM-Tris buffer with sodium citrate at an ionic strength of 1.2 to 1.6. Large hexagonal rods grew in the cells in several hours (Fig. 4). The same crystal form was also obtained using the technique of vapor diffusion with ammonium sulfate as precipitant. Again, large hexagonal rods grew in several hours. The crystallization conditions were refined using mierodialysis buttons to an optimal ionic strength of 0.8 in citrate at pH 8.0 in 50 m~-Tris, t{C1 buffer and a protein
L E T T E R S TO THE E D I T O R
ll03
Fic. 2. Type I quinolinate phosphoribosyltransferase crystals. Grown at 4°C in 50 raM-potassium phosphate buffer (pl-I 7.0), 14~o saturation ammonium sulfate and 23 mg quinolinate phosphoribosyltransferase/ml. concentration of 20 to 25 mg/ml. Under these conditions, the enzyme was completely soluble at 4°C but produced large hexagonal rods ( > 2 m m long) at room temperature in one to two weeks. I n addition to the hexagonal apoenzyme crystals, a pseudo-ternary complex with the identical morphology has been grown. This complex contains Mg 2+-phosphoribosyl pyrophosphate and the competitive inhibitor phthalic acid (Taguchi & Iwai, 1976). I f the molecule undergoes a conformational change (Paekman & Jakoby, 1965,1967; Mann & Byerrum, 1974) on binding this complex, the change does not alter the crystal morphology. The ape crystals were found to diffract to as far as 2"2 A resolution with sealed tube Cu radiation. The space group of this crystal form was subsequently identified as P6322 with a --~ b = 120.9 A, c -- 95-0 A. High resolution precession photographs (t* = 16°) of the hkO and hOl zones are shown in Figure 5. Assuming a molecular weight of 172,000, Vm values of 3.59, 2-39 and 1.79 A3/dalton are calculated for two, three and four molecules per unit cell, respectively. All of these ratios are within the acceptable range for protein crystals (Matthews, 1968). Since the space group has 12 equivalent positions, the unit cell will accommodate only two or four molecules in special positions. A consequence of locating the molecule in these positions is that it is necessarily hexameric. This is in contradiction to the five subunit model proposed by Taguehi & Iwai (1975a). Furthermore, since several different special positions are possible, the molecule m a y belong only to point s y m m e t r y group 3, 32 or 6. A two-molecule unit cell makes the crystallographic asymmetric unit one-
1101
W. D O N A L D L, M U S I C K
FIG. 3. /s = 2° precession photograph of type I quinolinate phosphoribosyltransferase taken perpendicular to the hexagonal face (hkO): a 2 h exposure on a 75 mm Buerger preecssion camera on an Elliott GX-6 rotating anode X-ray source (35 kV, 18 mA, Ni-filtered CuKa radiation). sixth of the quinolinate phosphoribosyltransferase molecule, whereas one-third of the molecule is the asymmetric unit in a four-molecule celt. However, the latter ease requires t h a t two molecules be placed along the z axis, allowing a maximal molecular dimension of 47 A in this direction. I f the quinolinate phosphoribosyltransferase molecule is approximated as a sphere of uniform density with a partial specific volume of 0.74 ml/g (Taguehi & Iwai, 1975a), a molecular diameter of 71 A is calculated. Since equilibrium eentrifugation studies have indicated t h a t the enzyme is a compact protein (Packman & J a k o b y , 1967; Taguchi & Iwai, 1975a), this approximation is justifiable. Therefore, a four-moleeule unit cell is an unlikely possibility. Furthermore, one of the two special positions allowed in a two-molecule unit cell also stacks two molecules on the z axis. Consequently, only the enantiomorphie positions (1/3, 2/3, 1/4; 2/3, 1/3, 3/4) or (2/3, 1/3, 1/4; 1/3, 2/3, 3/4) are reasonable locations for the molecule. These special positions require a molecular point s y m m e t r y of 32. An a t t e m p t to put these arguments on a more quantitative basis was made b y calculating intensities based on a model structure and comparing them to the observed low-order reflections. The model was a constant density sphere of variable radius
LETTERS
TO T H E E D I T O R
1105
FIG. 4. Type I I quinolinate phosphoribosyltransferase crystals. Grown at room temperature in 50 mN-Tris buffer (pit 8"0) at an ionic strength of 1.2 in citrate and 20 mg quinolinate phosphoribosyltransferase]ml.
placed in the special positions dictated by the two and four-molecule unit cells. The model which best fit the data contained two spheres of approximately 70 _£_diameter at centers 1/3, 2/3, 1/4 and 2/3, 1/3, 3/4. All models placing two molecules on the z axis poorly predicted the low-order intensities. A further refinement of the model was performed using a cluster of spheres with 32 symmetry at the indicated centers. The only constraint utilized was that the top trimerie aggregate of spheres be in contact with the bottom aggregate. The sphere radii were allowed to vary along with the radial separation of each trimerie aggregate. This model produced a better fit to the data with an optimal sphere radius of 19 A and a radial separation of 32 A. A diagram of this packing arrangement is shown in Figure 6. As seen in Figure 4, the hexad axis runs along the length of the crystal. The Laue symmetry of the lattice reduces the unique portion of reciprocal space to a 30 ° wedge along this axis. Consequently, this crystal form of quinolinate phosphoribosyltransferase is particularly well suited to the technique of oscillation photography and facilitates rapid data collection, which is now underway. A study of the amino acid sequence of quinolinate phosphoribosyltransferase has been started at Purdue University (3/[. Hermodson, personal communication) and thus far cyanogen bromide fragments as well as lysine and arginine-blocked tryptic peptides have been characterized. Preliminary sequencing studies of these fragments are now- in progress. 72
©
cD
®
~o
v
~
~,.~
~N ~
o
0 . ~
~
N
~.~
N
~
L E T T E R S TO T H E E D I T O R
1107 o
¼ FIG. 6. The packing arrangement of the quinolinate phosphoribosyltransferase, 32 hexamers deduced from low order reflections. I am grateful to Drs M. G. Rossmann, A. Sieignano and J. E. Johnson for helpful suggestions and comments, and to Sharon S. Wilder for help in the preparation of the manuscript. The work was supported by the National Institutes of Health (grant no. GM 10704) and the National Science Foundation (grant no. BMS74-23537). Departmertt of BiologieM Sciences Purdue University West Lafayette, Ind. 47907, U.S.A.
W . DONALD L. MUSlCK
]~eceived 3 August 1977 REFERENCES Ahmad, F. & Moat, A. G. (1966). J. Biol. Chem. 241,775 780. Gaertner, F. H. & Sherry, A. S. (1977). Biochim. Biophys. Aeta, 482, 453-460. Gholson, R. K. (1966). Nature (London), 212, 933-934. Isquith, A. J. & Moat, A. G. (1966). Biochem. Biophys. Res. Commun. 22, 565-571. Mann, D. F. & Byerrum, 1~. U. (1974). J. Biol. Chem. 249, 6817-6823. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Ogasawara, N., Chandler, J. L. 1~., Gholson, R. K., t~osser, R. J. & Andreoli, A. J. (1967). B~ochim. Biophys. Aeta, 141, 199-201. Ortega, ~ . V. & Brown, G. M. (1960). J. Biol. Chem. 235, 2939-2945. Packman, P. M. & Jakoby, W. B. (1965). Biochem. Biophys. Res. Commun. 18, 710-715. Packman, P. M. & Jakoby, W. B. (1967). J. Biol. Chem. 242, 2075-2079. Sakakibara, S., Wicks, F. D. & Gholson, 1~. K. (1977). Biochem. Biophys. Res. Commun. 76, 158-166. Scott, T. A., Bellion, E. & Mattey, M. (1969). Eur. J. Biochem. 10, 318-323. Taguchi, I~I. & Iwai, K. (1975a). Agr. BioL Chem. 39, 1493-1500. Taguehi, I-I. & Iwai, K. (1975b). Agr. Biol. Chem. 39, 1599-1604. Taguehi, H. & Iwai, K. (1976). Agr. Biol. Chem. 40, 385-389.