J. Mol. Biol. (1990) 216, 235-237
Initiating a Crystallographic Study of Trypanothione Reductase William N. Hunter~t, Keith Smith 2, Zygmunt Derewenda 3, Stephen J. Harrop ~ Jarjis Habash ~, M. S. Islam~:~, John R. Helliwell ~'4 and Alan H. Fairlamb 2 1Department of Chemistry, University of Manchester Oxford Road, Manchester M13 9PL, U.K. 2Department of Medical Parasitology, London School of Hygiene and Tropical Medicine Keppel Street, London WCIE 7HT, U.K. 3Department of Chemistry, University of York Heslington, York Y01 5DD, U.K. 4SERC Daresbury Laboratory Daresbury, Warrington, Cheshire WA4 4AD, U.K. (Received 19 July 1990; accepted 3 August 1990) We have obtained well-ordered single crystals of the flavoenzyme trypanothione reductase fi'om Crithidia fasciculata. The crystals are tetragonal rods with unit cell dimensions a - 128.6 A, c = 92.5 A. The diffraction pattern corresponds to a primitive lattice, Laue class 4/m. Diffraction to better than 2-4 A has been recorded at the Daresbury Synchrotron. The accurate elucidation of the three-dimensional structure of this enzyme is required to support the rational design of compounds active against a variety of tropical diseases caused by trypanosomal parasites.
Parasitic protozoa of the order Kinetoplastida are the causal agents of a wide range of tropical diseases. Among these are African sleeping sickness, Chagas' disease and Leishmaniasis. The treatment of these ailments is unsatisfactory and less toxic, more effective, drugs are being sought. A number of research groups are engaged in studying the metabolism of trypanosomes in an effort to identify viable biochemical targets for rational drug design (Fairlamb, 1989; Hol, 1986). One potential target is the glycosome, an organelle that contains the enzymes responsible for glycolysis in these organisms (Opperdoes, 1987). Several trypanosomal glycolytic enzymes are being characterized by a variety of methods including single crystal X-ray diffraction techniques (Hol et al., 1989). However, the problems of designing selective inhibitors are compounded by the high degree of homology between the substrate-binding sites of the mammalian host and parasite enzymes. An enzyme system unique to trypanosomes may afford a better chance of success in rational drug design. Such a system has been identified in trypanothione reducWe dedicate this paper to the memory of Dr G. B. Henderson. t Author to whom all correspondence should be addressed. :~On leave fl'om the Department of Physics, Rajshahi University, Bangladesh. 0022-2836/90/220235-03 $03.00/0
tase, an enzyme that occupies a pivotal role in essential metabolic functions of these parasites (Fairlamb & Henderson, 1987; Fairlamb, 1989). Mammalian cells use glutathione (~-glutamylcysteinyl-glycine; GSH§) in a diverse range of protective, regulatory and co-factor functions (Meister, 1989). Whilst maintaining a thiol-redox balance and in defence against oxidative damage, GSH is oxidized to the disulphide, GSSG. The regeneration of GSH is accomplished by the NADPH-dependent flavoenzyme, glutathione reductase. Trypanosomes do not contain glutathione reductase but accomplish the reduction of GSSG by means of an intermediate co-factor, trypanothione (N1,NS-bis(glutathionyl) spermidine: see Fairlamb et al., 1985). Intracellular trypanothione is maintained as the thiol by means of the NADPH-dependent flavoenzyme trypanothione reductase (Shames et al., 1986; Krauth-Siegel et al., 1987). This enzyme is similar to glutathione reductase in a number of ways: both enzymes function as dimers (the monomer is about M r 52,000), use FAD co-enzyme, use N A D P H as electron donor and appear to have similar reaction mechanisms (for a review, see Ghisla & Massey, 1989; Schirmer et al., 1989). The similarity extends to the amino acid § Abbreviation used: GSH, ?-glutamyl-eysteinylglycine. 235
© 1990 Academic Pl~ss Limited
236
W.N.
Hunter et al.
sequence where 60% homology is observed (Shames et al., 1988; Sullivan et al., 1989). However, both enzymes show mutually exclusive specificities for their respective disulphide substrates (Henderson et al., 1987; Shames et al., 1986). Our aim is to understand the molecular basis for these differences between the enzymes of host and parasite and to use this information in the design of new anti-trypanosomal agents. The human glutathione reductase structure has been determined and analysed thoroughly to a resolution of about 1"5 A (1 A = 0"l nm: see Karplus & Schulz, 1987, 1989). The refined co-ordinates for this structure have been deposited with the Brookhaven Protein Databankt (Bernstein et al., 1977). Access to these co-ordinates will aid our study. Trypanothione reductase from the insect parasite Crithidia fasciculata was isolated essentially as described by Shames et al. (1986) and we used 8 mg of this enzyme for a series of microcrystallization trials, typically using 4 to 5 gl of protein solution. Different techniques for achieving supersaturation of the enzyme solution were tried; they included the use of dialysis buttons, vapour diffusion with hanging drops and interface diffusion. A variety of precipitants, additions and several pH values were also tested at both 20 and 4°C. It was observed that the hanging drop method at the lower temperature gave better results than other techniques being applied and this then became the preferred method for subsequent crystallization trials. Once conditions for crystallization had been found the volume of the drops was increased to allow more protein in the conditions. This scaling up was necessary to obtain crystals large enough for diffraction experiments. The optimized conditions for reproducible growth of crystals involves equilibrating 20 #l of an 18mg enzyme/ml solution, 0-1 M-potassium phosphate (pH 7"0), 50% (w/v) saturated ammonium sulphate against an 80~o saturated ammonium sulphate solution by vapour diffusion using hanging drops. Crystals appeared after a period of weeks at 4°C. The crystals are well-ordered, yellow, tetragonal rods which grow on occasion to lengths in excess of l'0 mm and thickness of 0"2 ram. The colour is a consequence of bound FAD. To characterize the diffraction, crystals were mounted in glass capillaries and exposed to X-rays (50kV, 25mA from a fine focus tube, CuKa, )~ = 1-5418A) with a Xentronics/Nicolet imaging proportional counter. The calibration and experimental details for use of this system are given by Derewenda & Helliwell (1989). A partial data set to 2-7 A resolution has been measured on this instrument. The unit cell was identified using the autoindexing routine of the R E F I N E program (Howard eta/., 1987) applied to the first 40 ° of data and the axial lengths were subsequently refined to a = 128-6A, c = 92-5A. In total 84,621 observations of 20,805 unique reflections were recorded. t Accession number 3GRS.PDB.
They were scaled and merged together assuming tetragonal 4/m symmetry with the X E N G E N suite of programs (Howard et al., 1987). The merging R-factor for symmetry-related reflections was 11.7 ~/o. Analysis of intensities along the e axis indicates space group P41 or enantiomorph P43. Assuming one dimer of trypanothione reductase per asymmetric unit the specific volume Vm is 3"5 Aa/dalton, two dimers in the asymmetric unit results in a calculated Vm value of 1'8 A3/dalton. These values are at the extremes of the range normally observed for protein crystals (Matthews, 1968). Three monomers per asymmetric unit (1.5 dimers) results in a calculated Vm value of 2-5A3/dalton. Self-rotation function calculations are being carried out to locate elements of noncrystallographic symmetry. The preliminary results suggest a single non-crystallographic 2-fold axis that is commensurate with a dimer in the asymmetric unit. The sequence homology between trypanothione reductase and glutathione reductase, as discussed above, indicates that the method of molecular replacement (Rossmann, 1972) may allow for structure solution using the refined co-ordinates of human glutathione reductase (Karplus & Schulz, 1987) as the search model. We have also used the Daresbury Synchrotron for our study. Diffraction has been recorded at better than 2"4 A resolution on Stations 7'2 and 9"6 using wavelengths of 1"488 A and 0"895 A, respectively. The experimental configuration of these protein crystallography stations is described by Heiliwell et al. (1982, 1986). We note that the crystals are radiation-stable but on occasion have been susceptible to drying out quickly. In summary, we have obtained well-ordered, radiation-stable crystals of trypanothione reductase. These crystals offer the opportunity for a high resolution structural analysis of this trypanosomal enzyme. Work is in progress to extend this study to the analysis of enzyme-substrate and enzymeinhibitor crystals and to other crystal forms of trypanothione reductase. Our structural results will complement the biochemical and pharmaceutical studies directed towards design and testing of new compounds active against trypanosomes. We gratefully acknowledge the encouragement and interaction of Ken Douglas and co-workers in the Department of Pharmacy at Manchester University. Colin Jackson, Colin Nave, Andy Thompson and Miroslav Papiz of the Daresbury Laboratory are thanked for their excellent support. M.S.I. is supported by the Commonwealth Scholarship Commission in the United Kingdom. Our studies are funded by the Wellcome Trust, the National Institutes of Health, U.S.A. (AI21429), the Hasselblad Foundation (for provision of a scanner), the University of Manchester and the SERC (U.K.). References
Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E.F., Brice, M.D., Rogers, J.R., Kennard, 0., Shimanouchi, T. & Tasumi, M. (1977). Eur. J. Biochem. 80, 319-324.
Communications Derewenda, Z. & Helliwell, J. 1%. (1989). J. Appl. Crystallogr. 22, 123-137. Fairlamb, A. H. (1989). Parasitology, 99S, 93-112. Fairlamb, A. H. & Henderson, G. B. (1987). In Host-Parasite Cellular and Molecular Interactions In Protozoal Infections (Chang, K.-P. & Snary, D., eds), pp. 29-40, NATO ASI Series, Springer-Verlag, Berlin. Fairlamb, A. H., Blackburn, P., Ulrich, P., Chait, B. T. & Cerami, A. (1985). Science, 227, 1485-1487. Ghisla, S. K. & Massey, V. (1989). Eur. J. Biochem. 181, 1-17. Helliwell, J. 1%.,Greenough, T. J., Carr, P. D., 1%ule,S. A., Moore, P. 1%., Thompson, A.W. & Worgan, T.S. (1982). g. Phys. E. 5, 1363-1372. Helliwell, J. 1%., Papiz, M. Z., Glover, I. D., Habash, J., Thompson, A. W., Moore, P. 1%.,Harris, N., Croft, D. & Pantos, E. (1986). Nucl. Instrum. Methods. Phys. Res. A246, 617-623. Henderson, G. B., Fairlamb, A. H., Ulrich, P. & Cerami, A. (1987). Biochemistry, 26, 3023-3027. Hol, W. (1986}. Ange. Chemie. Intl. Ed. Engl. 25,767-778. Hol, W. G. J., Weirenga, R. K., Groendijk, H., Read, 1%.J., Thunnissen, A.M.W.H., Noble, M.E.M., Kalk, K. H., Vellieux, F. M. D., Opperdoes, F. 1%. & Michels, P. A. M. (1989). In Molecular Recognition: Chemical and Biochemical Problems (Roberts, S. M., ed.), pp. 84-93, 1%oyalSociety of Chemistry, London. Howard, A. J., Gilliland, G. L., Finzel, B. C., Poulos, T.L., Ohlendorf, D.H. & Salemme, F. 1%. (1987). Acta Crystallogr. 20, 383-387.
237
Karplus, P. A. & Schulz, G. E. (1987). J. Mol. Biol. 195, 701-729. Karplus, P. A. & Schulz, G. E. (1989). J. Mot. Biol. 210, 163-180. Krauth-Siegel, 1%. L., Enders, B., Henderson, G. B., Fairlamb, A.H. & Schirmer, R.H. (1987). Eur. J. Biochem. 164, 123-128. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Meister, A. (1989). In Glutathione; Chemical, Biochemical and Medical Aspects (Dolphin, D., Poulson, R. & Avramovic, O., eds), pp. 367-474, John Wiley & Sons, New York. Opperdoes, F. 1%. (1987). Annu. Rev. Microbiol. 41, 127-151. 1%ossmann, M. G. (1972). The Molecular Replacement Method, Gordon & Breach, New York. Schirmer, R. H., Krauth-Siegel, 1%. L. & Schulz, G. E. (1989). In Glutathione: Chemical, Biochemical and Medical Aspects (Dolphin, P., Poulson, R. & Avramovic, O., eds), part A, pp. 553-596, John Wiley & Sons, New York. Shames, S. L., Fairlamb, A. H., Cerami, A. & Walsh, C. T. (1986). Biochemistry, 25, 3519-3526. Shames, S. L., Kimmel, B. E., Peoples, O. P., Agabian, N. & Walsh, C. T. (1988). Biochemistry, 27, 5014-5019. Sullivan, F. X., Shames, S. L. & Walsh, C. T. (1989). Biochemistry, 28, 4986-4992.
Edited by A. Klug
Initiating a Crystallographic Study of Trypanothione Reductase William N. Hunter~t, Keith Smith 2, Zygmunt Derewenda 3, Stephen J. Harrop ~ Jarjis Habash ~, M. S. Islam~:~, John R. Helliwell ~'4 and Alan H. Fairlamb 2 1Department of Chemistry, University of Manchester Oxford Road, Manchester M13 9PL, U.K. 2Department of Medical Parasitology, London School of Hygiene and Tropical Medicine Keppel Street, London WCIE 7HT, U.K. 3Department of Chemistry, University of York Heslington, York Y01 5DD, U.K. 4SERC Daresbury Laboratory Daresbury, Warrington, Cheshire WA4 4AD, U.K. (Received 19 July 1990; accepted 3 August 1990) We have obtained well-ordered single crystals of the flavoenzyme trypanothione reductase fi'om Crithidia fasciculata. The crystals are tetragonal rods with unit cell dimensions a - 128.6 A, c = 92.5 A. The diffraction pattern corresponds to a primitive lattice, Laue class 4/m. Diffraction to better than 2-4 A has been recorded at the Daresbury Synchrotron. The accurate elucidation of the three-dimensional structure of this enzyme is required to support the rational design of compounds active against a variety of tropical diseases caused by trypanosomal parasites.
Parasitic protozoa of the order Kinetoplastida are the causal agents of a wide range of tropical diseases. Among these are African sleeping sickness, Chagas' disease and Leishmaniasis. The treatment of these ailments is unsatisfactory and less toxic, more effective, drugs are being sought. A number of research groups are engaged in studying the metabolism of trypanosomes in an effort to identify viable biochemical targets for rational drug design (Fairlamb, 1989; Hol, 1986). One potential target is the glycosome, an organelle that contains the enzymes responsible for glycolysis in these organisms (Opperdoes, 1987). Several trypanosomal glycolytic enzymes are being characterized by a variety of methods including single crystal X-ray diffraction techniques (Hol et al., 1989). However, the problems of designing selective inhibitors are compounded by the high degree of homology between the substrate-binding sites of the mammalian host and parasite enzymes. An enzyme system unique to trypanosomes may afford a better chance of success in rational drug design. Such a system has been identified in trypanothione reducWe dedicate this paper to the memory of Dr G. B. Henderson. t Author to whom all correspondence should be addressed. :~On leave fl'om the Department of Physics, Rajshahi University, Bangladesh. 0022-2836/90/220235-03 $03.00/0
tase, an enzyme that occupies a pivotal role in essential metabolic functions of these parasites (Fairlamb & Henderson, 1987; Fairlamb, 1989). Mammalian cells use glutathione (~-glutamylcysteinyl-glycine; GSH§) in a diverse range of protective, regulatory and co-factor functions (Meister, 1989). Whilst maintaining a thiol-redox balance and in defence against oxidative damage, GSH is oxidized to the disulphide, GSSG. The regeneration of GSH is accomplished by the NADPH-dependent flavoenzyme, glutathione reductase. Trypanosomes do not contain glutathione reductase but accomplish the reduction of GSSG by means of an intermediate co-factor, trypanothione (N1,NS-bis(glutathionyl) spermidine: see Fairlamb et al., 1985). Intracellular trypanothione is maintained as the thiol by means of the NADPH-dependent flavoenzyme trypanothione reductase (Shames et al., 1986; Krauth-Siegel et al., 1987). This enzyme is similar to glutathione reductase in a number of ways: both enzymes function as dimers (the monomer is about M r 52,000), use FAD co-enzyme, use N A D P H as electron donor and appear to have similar reaction mechanisms (for a review, see Ghisla & Massey, 1989; Schirmer et al., 1989). The similarity extends to the amino acid § Abbreviation used: GSH, ?-glutamyl-eysteinylglycine. 235
© 1990 Academic Pl~ss Limited
236
W.N.
Hunter et al.
sequence where 60% homology is observed (Shames et al., 1988; Sullivan et al., 1989). However, both enzymes show mutually exclusive specificities for their respective disulphide substrates (Henderson et al., 1987; Shames et al., 1986). Our aim is to understand the molecular basis for these differences between the enzymes of host and parasite and to use this information in the design of new anti-trypanosomal agents. The human glutathione reductase structure has been determined and analysed thoroughly to a resolution of about 1"5 A (1 A = 0"l nm: see Karplus & Schulz, 1987, 1989). The refined co-ordinates for this structure have been deposited with the Brookhaven Protein Databankt (Bernstein et al., 1977). Access to these co-ordinates will aid our study. Trypanothione reductase from the insect parasite Crithidia fasciculata was isolated essentially as described by Shames et al. (1986) and we used 8 mg of this enzyme for a series of microcrystallization trials, typically using 4 to 5 gl of protein solution. Different techniques for achieving supersaturation of the enzyme solution were tried; they included the use of dialysis buttons, vapour diffusion with hanging drops and interface diffusion. A variety of precipitants, additions and several pH values were also tested at both 20 and 4°C. It was observed that the hanging drop method at the lower temperature gave better results than other techniques being applied and this then became the preferred method for subsequent crystallization trials. Once conditions for crystallization had been found the volume of the drops was increased to allow more protein in the conditions. This scaling up was necessary to obtain crystals large enough for diffraction experiments. The optimized conditions for reproducible growth of crystals involves equilibrating 20 #l of an 18mg enzyme/ml solution, 0-1 M-potassium phosphate (pH 7"0), 50% (w/v) saturated ammonium sulphate against an 80~o saturated ammonium sulphate solution by vapour diffusion using hanging drops. Crystals appeared after a period of weeks at 4°C. The crystals are well-ordered, yellow, tetragonal rods which grow on occasion to lengths in excess of l'0 mm and thickness of 0"2 ram. The colour is a consequence of bound FAD. To characterize the diffraction, crystals were mounted in glass capillaries and exposed to X-rays (50kV, 25mA from a fine focus tube, CuKa, )~ = 1-5418A) with a Xentronics/Nicolet imaging proportional counter. The calibration and experimental details for use of this system are given by Derewenda & Helliwell (1989). A partial data set to 2-7 A resolution has been measured on this instrument. The unit cell was identified using the autoindexing routine of the R E F I N E program (Howard eta/., 1987) applied to the first 40 ° of data and the axial lengths were subsequently refined to a = 128-6A, c = 92-5A. In total 84,621 observations of 20,805 unique reflections were recorded. t Accession number 3GRS.PDB.
They were scaled and merged together assuming tetragonal 4/m symmetry with the X E N G E N suite of programs (Howard et al., 1987). The merging R-factor for symmetry-related reflections was 11.7 ~/o. Analysis of intensities along the e axis indicates space group P41 or enantiomorph P43. Assuming one dimer of trypanothione reductase per asymmetric unit the specific volume Vm is 3"5 Aa/dalton, two dimers in the asymmetric unit results in a calculated Vm value of 1'8 A3/dalton. These values are at the extremes of the range normally observed for protein crystals (Matthews, 1968). Three monomers per asymmetric unit (1.5 dimers) results in a calculated Vm value of 2-5A3/dalton. Self-rotation function calculations are being carried out to locate elements of noncrystallographic symmetry. The preliminary results suggest a single non-crystallographic 2-fold axis that is commensurate with a dimer in the asymmetric unit. The sequence homology between trypanothione reductase and glutathione reductase, as discussed above, indicates that the method of molecular replacement (Rossmann, 1972) may allow for structure solution using the refined co-ordinates of human glutathione reductase (Karplus & Schulz, 1987) as the search model. We have also used the Daresbury Synchrotron for our study. Diffraction has been recorded at better than 2"4 A resolution on Stations 7'2 and 9"6 using wavelengths of 1"488 A and 0"895 A, respectively. The experimental configuration of these protein crystallography stations is described by Heiliwell et al. (1982, 1986). We note that the crystals are radiation-stable but on occasion have been susceptible to drying out quickly. In summary, we have obtained well-ordered, radiation-stable crystals of trypanothione reductase. These crystals offer the opportunity for a high resolution structural analysis of this trypanosomal enzyme. Work is in progress to extend this study to the analysis of enzyme-substrate and enzymeinhibitor crystals and to other crystal forms of trypanothione reductase. Our structural results will complement the biochemical and pharmaceutical studies directed towards design and testing of new compounds active against trypanosomes. We gratefully acknowledge the encouragement and interaction of Ken Douglas and co-workers in the Department of Pharmacy at Manchester University. Colin Jackson, Colin Nave, Andy Thompson and Miroslav Papiz of the Daresbury Laboratory are thanked for their excellent support. M.S.I. is supported by the Commonwealth Scholarship Commission in the United Kingdom. Our studies are funded by the Wellcome Trust, the National Institutes of Health, U.S.A. (AI21429), the Hasselblad Foundation (for provision of a scanner), the University of Manchester and the SERC (U.K.). References
Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E.F., Brice, M.D., Rogers, J.R., Kennard, 0., Shimanouchi, T. & Tasumi, M. (1977). Eur. J. Biochem. 80, 319-324.
Communications Derewenda, Z. & Helliwell, J. 1%. (1989). J. Appl. Crystallogr. 22, 123-137. Fairlamb, A. H. (1989). Parasitology, 99S, 93-112. Fairlamb, A. H. & Henderson, G. B. (1987). In Host-Parasite Cellular and Molecular Interactions In Protozoal Infections (Chang, K.-P. & Snary, D., eds), pp. 29-40, NATO ASI Series, Springer-Verlag, Berlin. Fairlamb, A. H., Blackburn, P., Ulrich, P., Chait, B. T. & Cerami, A. (1985). Science, 227, 1485-1487. Ghisla, S. K. & Massey, V. (1989). Eur. J. Biochem. 181, 1-17. Helliwell, J. 1%.,Greenough, T. J., Carr, P. D., 1%ule,S. A., Moore, P. 1%., Thompson, A.W. & Worgan, T.S. (1982). g. Phys. E. 5, 1363-1372. Helliwell, J. 1%., Papiz, M. Z., Glover, I. D., Habash, J., Thompson, A. W., Moore, P. 1%.,Harris, N., Croft, D. & Pantos, E. (1986). Nucl. Instrum. Methods. Phys. Res. A246, 617-623. Henderson, G. B., Fairlamb, A. H., Ulrich, P. & Cerami, A. (1987). Biochemistry, 26, 3023-3027. Hol, W. (1986}. Ange. Chemie. Intl. Ed. Engl. 25,767-778. Hol, W. G. J., Weirenga, R. K., Groendijk, H., Read, 1%.J., Thunnissen, A.M.W.H., Noble, M.E.M., Kalk, K. H., Vellieux, F. M. D., Opperdoes, F. 1%. & Michels, P. A. M. (1989). In Molecular Recognition: Chemical and Biochemical Problems (Roberts, S. M., ed.), pp. 84-93, 1%oyalSociety of Chemistry, London. Howard, A. J., Gilliland, G. L., Finzel, B. C., Poulos, T.L., Ohlendorf, D.H. & Salemme, F. 1%. (1987). Acta Crystallogr. 20, 383-387.
237
Karplus, P. A. & Schulz, G. E. (1987). J. Mol. Biol. 195, 701-729. Karplus, P. A. & Schulz, G. E. (1989). J. Mot. Biol. 210, 163-180. Krauth-Siegel, 1%. L., Enders, B., Henderson, G. B., Fairlamb, A.H. & Schirmer, R.H. (1987). Eur. J. Biochem. 164, 123-128. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Meister, A. (1989). In Glutathione; Chemical, Biochemical and Medical Aspects (Dolphin, D., Poulson, R. & Avramovic, O., eds), pp. 367-474, John Wiley & Sons, New York. Opperdoes, F. 1%. (1987). Annu. Rev. Microbiol. 41, 127-151. 1%ossmann, M. G. (1972). The Molecular Replacement Method, Gordon & Breach, New York. Schirmer, R. H., Krauth-Siegel, 1%. L. & Schulz, G. E. (1989). In Glutathione: Chemical, Biochemical and Medical Aspects (Dolphin, P., Poulson, R. & Avramovic, O., eds), part A, pp. 553-596, John Wiley & Sons, New York. Shames, S. L., Fairlamb, A. H., Cerami, A. & Walsh, C. T. (1986). Biochemistry, 25, 3519-3526. Shames, S. L., Kimmel, B. E., Peoples, O. P., Agabian, N. & Walsh, C. T. (1988). Biochemistry, 27, 5014-5019. Sullivan, F. X., Shames, S. L. & Walsh, C. T. (1989). Biochemistry, 28, 4986-4992.
Edited by A. Klug
