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Preliminary crystallographic data for adenylosuccinate synthetase from rabbit skeletal muscle
.J. Mol. Hid. (1979) 132, 729-731
LETTERS
TO THE EDITOR
Preliminary Crystallographic Data for Adenylosuccinate Synthetase from Rabbit Skeletal Muscle Sir~gl(b crystals of aderlylosllccirlate synthetase, an essential compor~r?~ll~of purilW I~iosyr~ttwsis, clxtracted from rabbit skeletal muscle were prepared as suit&It, sl)e:‘:irnr~ns for X -ra.y struct.ure analysis. Tlto crystal kwdongs to ttw space group /‘4,$,% or 1’&Z12 wittl unit cell dimensions a = b = 71.2 .!L, c : 194.8 A. l‘tlt~ ;isylrunr%ric? Iinit, contains one protein molecule of 64,000 molecular weight. At)tention ha.s been focused recently on adenylosuccinate synthetase (IMP : I,aspartate ligase (GDP), EC 6.3.4.4) catalyzing the formation of adenylosuccinat’c from IMP and a.spartat,e in the presence of GTP, because this enzyme reaction is on >I branch-point of the main pathway of the purine nucleotide biosynthesis. and on t,he purine nucleotide cycle (Fig. I), which is possibly involved in ammoniagenesis ilnd glycolysis (Lowenstein, 1972). Cti-COOH I
II
CH*--COOH
I Cl-l -COOH ribW-
Ai ASS GTP a
I f-J’-‘,
(Asp)
(AMP)
l’rc:. 1. Thn
waotion
catalyzed
by
COOH
NH
N&CyN\ I HC\N/C,N ” /
phosphote (IMP)
-
f” I
Cibosephosphate
adenylosuccinate synthetane (ASS) ant1 the purinr nucleotitl~~
C:VClO.
‘I’ht, t,WJ types of adenylosuccinate synthetase have been found in rat liver, the M type showing immunological cross-reactivity with the muscle enzyme, and t.he 1, t’,vpe which does not have such an activity (Matsuda et al.: 1977). The M type enzyme in the liver and t,he muscle enzyme were more susceptible to fructose 1 ,(i-diphosphat,r inhibition than the L type enzyme: while the latter was more susceptible to mononucleotide inhibition than the former. On the hasis of these facts: an hypothesis was proposed on the action of these enzyme; that is. the L type enzyme might be preferable on the other hand, the M t’ype for the regulation of purine nucleotide biosynthesis; clnzyme or the muscle enzyme might be preferable for t’he regulat,ion of ammoniagenesis and glycolgsis. Furthermore, the latter two enzymes were bound to F-a&in (Ogat\la et al., 1978). Tn addition, AMP deaminase (AMP aminohydrolase, EC 3.5.4.6).
S. HARAIIA
730
El
AL.
another component enzyme of the purine nucleotide cycle, was also bound to myosin (Shiraki et al., 1979). This classification of adenylosuccinate synthetase and AMP deaminase in muscle contractile proteins gives a, firm basis for the hypothesis ment,ioned above, especially in skeletal muscle. The structure analysis of this protein seems to reveal some keys to elucidate the difference in the regulatory properties of these enzymes on a structural basis. The crystals of adenylosuccinat,e synthetase from rabbit skeletal muscle (Muirhead & Bishop, 1974), and from rat skeletal muscle (Ogawa et al., 1977) have been obtained; however, they were too small for X-ray experiments. The present paper is concerned with the preparation of large single crystals and the preliminary X-ray crystallographic data of the enzyme. The enzyme was isolated from rabbit skeletal muscle (about 8 kg) essentially by the method of Muirhead & Bishop (1974) yielding approxima,tely 80 mg of enzyme. A preliminary search for conditions favouring the growth of single crystals suitable for X-ray structural analysis was carried out by both dialysis and batch crystallization methods. Polyethylene glycol (M, = 1000 and 6000) and Z-methyl-2,4pentanediol were used as precipitants in the latter method. But polyethylene glycol did not give good crystals and the methylpentanediol only precipitated amorphous protein. Good crystals were obtained by dialysis against 6 m&I-sodium ph0sphat.e (pH 7.0) containing 1 mM-dithiothreitol, I miw-Mg2+ and 60(:{, sat’urated ammonium sulphate using microdialysis buttons in a 4°C cold room. The concentration of prot,ein was 1.79; (w/v) and small diamond-shaped crystals (about 0.3 mm in t,he largest dimension) were obtained after three months. Further attempts to obta,in single large adenylosuccinate synthetase crystals were carried out’ in the same buffer at 4°C: suit)able conditions for t’he growth of large crystals were shown to be 42 t’o 44 y,b saturated ammonium sulphat’e and a protein concentration of 4q; (w/v). IJnder these conditions crystals grew to approximately 1.0 mm across and 0.3 mm in thickness after three months. For X-ray diffraction experiments, a crystal was mounted in a thin-walled glass capillary in the usual way and diffraction patterns of the (hk0) and (Okl) zones were recorded with a Buerger precession camera (Xi-filbered CuKa radiation, crystal-tofilm distance of 60 mm). The crystals are tetragonal with unit cell dimensions of a := h = 71.2 & (: = 194.8 A and the space group is P4,2,2 or P4,2,2. Assuming that the asymmetric unit contains one protein molecule, the> I’, value (Matt,hews. 1968) is 2.29 &d a It on and this value lies within the range usually found for proteins. Crystallization under the same conditions but with GMP, inhibitory to enzymatic activity, also gave good crystals. Further experiments with AT-formyl-hydroxyinhibitor with L-aspartate. aminoacetate (Shigeura, 1963), which is a competitive and a search for good heavy-atom derivat,ives are now underway. Institute for Protein Osaka University Suita 565, Japan
Received
10 April
Research
SHICEHARU HARADA MATSIJVRA NOBUO TANAKA MASAO KAKUDO YOSHIHIRO MATSUDA HIROHUMI OGAWA HIROSHI SHIRARI HACHIRO NAKAGAWA YOSHIKI
1979
LETTERS
TO THE
EDITOR
73 I
REFERENCES Lowerlstri[r, J. M. (1972). Physiol. Rev. 52, 382-414. Matsuda, Y., Ogawa, H., Fukutome, S., Shiraki, H. & Nakagawa. H. (1977). Hiochettt Biophys. Res. Commun.. 78, 766-771. Mat>theus. 13. W. (1968). .I. Mol. Bid. 33, 491-497. Muirhtiad, K. M. & Rishop, S. H. (1974). J. Riol. Ghem. 25, 459 464. Ogawa. H.. StGraki, H., Mat)sllda, Y. & Nakagawa, H. (1977). ,I. Biochem. 81, 8.59~~869. Oynua, H.. Shiraki, H., Matsuda, Y. & Nakagawa, H. (1978). Eur. J. B&hem. 85, 331~ 337. Slrigeura, H. 7’. (1963). J. Biol. Chem. 12, 3999-4001. Shiraki. H., Ogaua, H., Matsuda, Y. & Nakagawa, H. (1979). Riochim. Hiophys. Acta, 566, 335 -344.
LETTERS
TO THE EDITOR
Preliminary Crystallographic Data for Adenylosuccinate Synthetase from Rabbit Skeletal Muscle Sir~gl(b crystals of aderlylosllccirlate synthetase, an essential compor~r?~ll~of purilW I~iosyr~ttwsis, clxtracted from rabbit skeletal muscle were prepared as suit&It, sl)e:‘:irnr~ns for X -ra.y struct.ure analysis. Tlto crystal kwdongs to ttw space group /‘4,$,% or 1’&Z12 wittl unit cell dimensions a = b = 71.2 .!L, c : 194.8 A. l‘tlt~ ;isylrunr%ric? Iinit, contains one protein molecule of 64,000 molecular weight. At)tention ha.s been focused recently on adenylosuccinate synthetase (IMP : I,aspartate ligase (GDP), EC 6.3.4.4) catalyzing the formation of adenylosuccinat’c from IMP and a.spartat,e in the presence of GTP, because this enzyme reaction is on >I branch-point of the main pathway of the purine nucleotide biosynthesis. and on t,he purine nucleotide cycle (Fig. I), which is possibly involved in ammoniagenesis ilnd glycolysis (Lowenstein, 1972). Cti-COOH I
II
CH*--COOH
I Cl-l -COOH ribW-
Ai ASS GTP a
I f-J’-‘,
(Asp)
(AMP)
l’rc:. 1. Thn
waotion
catalyzed
by
COOH
NH
N&CyN\ I HC\N/C,N ” /
phosphote (IMP)
-
f” I
Cibosephosphate
adenylosuccinate synthetane (ASS) ant1 the purinr nucleotitl~~
C:VClO.
‘I’ht, t,WJ types of adenylosuccinate synthetase have been found in rat liver, the M type showing immunological cross-reactivity with the muscle enzyme, and t.he 1, t’,vpe which does not have such an activity (Matsuda et al.: 1977). The M type enzyme in the liver and t,he muscle enzyme were more susceptible to fructose 1 ,(i-diphosphat,r inhibition than the L type enzyme: while the latter was more susceptible to mononucleotide inhibition than the former. On the hasis of these facts: an hypothesis was proposed on the action of these enzyme; that is. the L type enzyme might be preferable on the other hand, the M t’ype for the regulation of purine nucleotide biosynthesis; clnzyme or the muscle enzyme might be preferable for t’he regulat,ion of ammoniagenesis and glycolgsis. Furthermore, the latter two enzymes were bound to F-a&in (Ogat\la et al., 1978). Tn addition, AMP deaminase (AMP aminohydrolase, EC 3.5.4.6).
S. HARAIIA
730
El
AL.
another component enzyme of the purine nucleotide cycle, was also bound to myosin (Shiraki et al., 1979). This classification of adenylosuccinate synthetase and AMP deaminase in muscle contractile proteins gives a, firm basis for the hypothesis ment,ioned above, especially in skeletal muscle. The structure analysis of this protein seems to reveal some keys to elucidate the difference in the regulatory properties of these enzymes on a structural basis. The crystals of adenylosuccinat,e synthetase from rabbit skeletal muscle (Muirhead & Bishop, 1974), and from rat skeletal muscle (Ogawa et al., 1977) have been obtained; however, they were too small for X-ray experiments. The present paper is concerned with the preparation of large single crystals and the preliminary X-ray crystallographic data of the enzyme. The enzyme was isolated from rabbit skeletal muscle (about 8 kg) essentially by the method of Muirhead & Bishop (1974) yielding approxima,tely 80 mg of enzyme. A preliminary search for conditions favouring the growth of single crystals suitable for X-ray structural analysis was carried out by both dialysis and batch crystallization methods. Polyethylene glycol (M, = 1000 and 6000) and Z-methyl-2,4pentanediol were used as precipitants in the latter method. But polyethylene glycol did not give good crystals and the methylpentanediol only precipitated amorphous protein. Good crystals were obtained by dialysis against 6 m&I-sodium ph0sphat.e (pH 7.0) containing 1 mM-dithiothreitol, I miw-Mg2+ and 60(:{, sat’urated ammonium sulphate using microdialysis buttons in a 4°C cold room. The concentration of prot,ein was 1.79; (w/v) and small diamond-shaped crystals (about 0.3 mm in t,he largest dimension) were obtained after three months. Further attempts to obta,in single large adenylosuccinate synthetase crystals were carried out’ in the same buffer at 4°C: suit)able conditions for t’he growth of large crystals were shown to be 42 t’o 44 y,b saturated ammonium sulphat’e and a protein concentration of 4q; (w/v). IJnder these conditions crystals grew to approximately 1.0 mm across and 0.3 mm in thickness after three months. For X-ray diffraction experiments, a crystal was mounted in a thin-walled glass capillary in the usual way and diffraction patterns of the (hk0) and (Okl) zones were recorded with a Buerger precession camera (Xi-filbered CuKa radiation, crystal-tofilm distance of 60 mm). The crystals are tetragonal with unit cell dimensions of a := h = 71.2 & (: = 194.8 A and the space group is P4,2,2 or P4,2,2. Assuming that the asymmetric unit contains one protein molecule, the> I’, value (Matt,hews. 1968) is 2.29 &d a It on and this value lies within the range usually found for proteins. Crystallization under the same conditions but with GMP, inhibitory to enzymatic activity, also gave good crystals. Further experiments with AT-formyl-hydroxyinhibitor with L-aspartate. aminoacetate (Shigeura, 1963), which is a competitive and a search for good heavy-atom derivat,ives are now underway. Institute for Protein Osaka University Suita 565, Japan
Received
10 April
Research
SHICEHARU HARADA MATSIJVRA NOBUO TANAKA MASAO KAKUDO YOSHIHIRO MATSUDA HIROHUMI OGAWA HIROSHI SHIRARI HACHIRO NAKAGAWA YOSHIKI
1979
LETTERS
TO THE
EDITOR
73 I
REFERENCES Lowerlstri[r, J. M. (1972). Physiol. Rev. 52, 382-414. Matsuda, Y., Ogawa, H., Fukutome, S., Shiraki, H. & Nakagawa. H. (1977). Hiochettt Biophys. Res. Commun.. 78, 766-771. Mat>theus. 13. W. (1968). .I. Mol. Bid. 33, 491-497. Muirhtiad, K. M. & Rishop, S. H. (1974). J. Riol. Ghem. 25, 459 464. Ogawa. H.. StGraki, H., Mat)sllda, Y. & Nakagawa, H. (1977). ,I. Biochem. 81, 8.59~~869. Oynua, H.. Shiraki, H., Matsuda, Y. & Nakagawa, H. (1978). Eur. J. B&hem. 85, 331~ 337. Slrigeura, H. 7’. (1963). J. Biol. Chem. 12, 3999-4001. Shiraki. H., Ogaua, H., Matsuda, Y. & Nakagawa, H. (1979). Riochim. Hiophys. Acta, 566, 335 -344.