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• W2000537633 abstract "Human cystathionine β-synthase is a pyridoxal 5′-phosphate enzyme containing a heme binding domain and an S-adenosyl-l-methionine regulatory site. We have investigated by single crystal microspectrophotometry the functional properties of a mutant lacking the S-adenosylmethionine binding domain. Polarized absorption spectra indicate that oxidized and reduced hemes are reversibly formed. Exposure of the reduced form of enzyme crystals to carbon monoxide led to the complete release of the heme moiety. This process, which takes place reversibly and without apparent crystal damage, facilitates the preparation of a heme-free human enzyme. The heme-free enzyme crystals exhibited polarized absorption spectra typical of a pyridoxal 5′-phosphate-dependent protein. The exposure of these crystals to increasing concentrations of the natural substratel-serine readily led to the formation of the key catalytic intermediate α-aminoacrylate. The dissociation constant ofl-serine was found to be 6 mm, close to that determined in solution. The amount of the α-aminoacrylate Schiff base formed in the presence of l-serine was pH independent between 6 and 9. However, the rate of the disappearance of the α-aminoacrylate, likely forming pyruvate and ammonia, was found to increase at pH values higher than 8. Finally, in the presence of homocysteine the α-aminoacrylate-enzyme absorption band readily disappears with the concomitant formation of the absorption band of the internal aldimine, indicating that cystathionine β-synthase crystals catalyze both β-elimination and β-replacement reactions. Taken together, these findings demonstrate that the heme moiety is not directly involved in the condensation reaction catalyzed by cystathionine β-synthase. Human cystathionine β-synthase is a pyridoxal 5′-phosphate enzyme containing a heme binding domain and an S-adenosyl-l-methionine regulatory site. We have investigated by single crystal microspectrophotometry the functional properties of a mutant lacking the S-adenosylmethionine binding domain. Polarized absorption spectra indicate that oxidized and reduced hemes are reversibly formed. Exposure of the reduced form of enzyme crystals to carbon monoxide led to the complete release of the heme moiety. This process, which takes place reversibly and without apparent crystal damage, facilitates the preparation of a heme-free human enzyme. The heme-free enzyme crystals exhibited polarized absorption spectra typical of a pyridoxal 5′-phosphate-dependent protein. The exposure of these crystals to increasing concentrations of the natural substratel-serine readily led to the formation of the key catalytic intermediate α-aminoacrylate. The dissociation constant ofl-serine was found to be 6 mm, close to that determined in solution. The amount of the α-aminoacrylate Schiff base formed in the presence of l-serine was pH independent between 6 and 9. However, the rate of the disappearance of the α-aminoacrylate, likely forming pyruvate and ammonia, was found to increase at pH values higher than 8. Finally, in the presence of homocysteine the α-aminoacrylate-enzyme absorption band readily disappears with the concomitant formation of the absorption band of the internal aldimine, indicating that cystathionine β-synthase crystals catalyze both β-elimination and β-replacement reactions. Taken together, these findings demonstrate that the heme moiety is not directly involved in the condensation reaction catalyzed by cystathionine β-synthase. cystathionine β-synthase pyridoxal 5′-phosphate polyethylene glycol High plasmatic levels of homocysteine have recently been associated with an increased risk of cardiovascular disease (1Refsum H. Ueland P. Nygard O. Vollset S.E. Annu. Rev. Med. 1998; 49: 31-62Crossref PubMed Scopus (1832) Google Scholar). Homocysteine is formed from S-adenosylhomocysteine and is either removed by cystathionine β-synthase (EC 4.2.1.22, CBS)1 in the trans-sulfuration pathway or remethylated to methionine in the methionine cycle. Deficiency of CBS is the major cause of inherited homocystinuria. CBS is a pyridoxal 5′-phosphate (PLP)-dependent enzyme catalyzing the synthesis of cystathionine from homocysteine and l-serine. The reaction proceeds via a β-replacement mechanism, similar to that of tryptophan synthase and O-acetylserine sulfhydrylase (2Borcsok E. Abeles R.H. Arch. Biochem. Biophys. 1982; 213: 695-707Crossref PubMed Scopus (54) Google Scholar). These enzymes belong to the β-family and fold II type within the PLP-dependent enzymes classification (3Alexander F.W. Sandmeier E. Metha P.K. Christen P. Eur. J. Biochem. 1994; 291: 953-960Crossref Scopus (343) Google Scholar, 4Grishin N.V. Phillips M.A. Goldsmith E.J. Protein Sci. 1995; 4: 1291-1304Crossref PubMed Scopus (339) Google Scholar). Other members of the β-family are serine and threonine dehydratases. The human CBS is a 63-kDa homotetramer containing one PLP and one heme per subunit (5Kery V. Bukovska G. Kraus J.P. J. Biol. Chem. 1994; 269: 25283-25288Abstract Full Text PDF PubMed Google Scholar). Whereas the functional role of PLP is known, the role of the heme is less clear. It was demonstrated that the heme redox state affects the affinity of the enzyme for the substrates (6Taoka S. Ohja S. Shan X.Y. Kruger W.D. Banerjee R. J. Biol. Chem. 1998; 273: 25179-25184Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Moreover, the catalytic activity of CBS is controlled by S-adenosylmethionine, which specifically binds to a C-terminal site. The trypsinolysis of a 18 kDa C-terminal fragment leads to a dimeric form, 2-fold more active than the native species and no longer regulated by S-adenosylmethionine (7Kery V. Poneleit L. Kraus J.P. Arch. Biochem. Biophys. 1998; 355: 222-232Crossref PubMed Scopus (138) Google Scholar). Similar results have been obtained on other truncated forms of CBS, obtained by insertion of nonsense mutations (8Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38: 13155-13161Crossref PubMed Scopus (82) Google Scholar). Investigation of the catalytic reaction brought about by PLP is complicated by the overlapping chromophoric properties of the heme. The recent investigation of the yeast enzyme, which does not contain heme groups, permitted a better characterization of the PLP role in the catalytic steps (9Jhee K.H. McPhie P. Miles E.W. J. Biol. Chem. 2000; 275: 11541-11544Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 10Jhee K.H. McPhie P. Miles E.W. Biochemistry. 2000; 39: 10548-10556Crossref PubMed Scopus (81) Google Scholar). An α-aminoacrylate species, absorbing at 470 and 320 nm, was observed upon reaction with l-serine. The nucleophilic attack of homocysteine on the α-aminoacrylate led to the formation of the product cystathionine. However, it is not yet known how closely the functional properties of the yeast enzyme resemble those of the human source.Structural studies of catalytic intermediates of tryptophan synthase and O-acetylserine sulfhydrylase (11Miles E.W. Adv. Enzymol. Relat. Areas Mol. Biol. 1991; 64: 93-172PubMed Google Scholar, 12Cook P.F. Hara S. Nalabolu S. Schnackerz K.D. Biochemistry. 1992; 31: 2298-2303Crossref PubMed Scopus (62) Google Scholar, 13Hyde C.C. Ahmed S.A. Padlan E.A. Miles E.W. Davies D.R. J. Biol. Chem. 1988; 263: 17857-17871Abstract Full Text PDF PubMed Google Scholar, 14Burkhard P. Rao G.S. Hohenester E. Schnackerz K.D. Cook P.F. Jansonius J.N. J. Mol. Biol. 1998; 283: 121-133Crossref PubMed Scopus (179) Google Scholar, 15Schneider T.R. Gerhardt E. Lee M. Liang P.H. Anderson K.S. Schlichting I. Biochemistry. 1998; 37: 5394-5406Crossref PubMed Scopus (134) Google Scholar, 16Rhee S. Parris K.D. Hyde C.C. Ahmed S.A. Miles E.W. Davies D.R. Biochemistry. 1997; 36: 7664-7680Crossref PubMed Scopus (143) Google Scholar, 17Rhee S. Miles E.W. Mozzarelli A. Davies D.R. Biochemistry. 1998; 37: 10653-10659Crossref PubMed Scopus (40) Google Scholar, 18Burkhard P. Tai C.H. Ristroph C.M. Cook P.F. Jansonius J.N. J. Mol. Biol. 1999; 291: 941-953Crossref PubMed Scopus (119) Google Scholar) have unveiled the nature of enzyme action. A common feature is an open to closed conformational transition of the active site taking place along the catalytic pathway. To fully exploit the structural information as well as to determine the structure of as many as possible catalytic intermediates, it is of paramount relevance to investigate the functional properties of the enzyme in the crystalline state by polarized absorption microspectrophotometry (19Mozzarelli A. Rossi G.L. Annu. Rev. Biophys. Biomol. Struct. 1996; 25: 343-365Crossref PubMed Scopus (104) Google Scholar). This approach was pioneered in the late sixties by Rossi and Bernhard (20Rossi G.L. Bernhard S.A. J. Mol. Biol. 1970; 49: 85-91Crossref PubMed Scopus (37) Google Scholar). In the case of tryptophan synthase (17Rhee S. Miles E.W. Mozzarelli A. Davies D.R. Biochemistry. 1998; 37: 10653-10659Crossref PubMed Scopus (40) Google Scholar, 21Mozzarelli A. Peracchi A. Rossi G.L. Ahmed S.A. Miles E.W. J. Biol. Chem. 1989; 264: 15774-15780Abstract Full Text PDF PubMed Google Scholar, 22Mozzarelli A. Peracchi A. Rovegno B. Dale G. Rossi G.L. Dunn M.F. J. Biol. Chem. 2000; 275: 6956-6962Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 23Peracchi A. Mozzarelli A. Rossi G.L. Biochemistry. 1995; 34: 9459-9465Crossref PubMed Scopus (78) Google Scholar) and O-acetylserine sulfhydrylase (24Mozzarelli A. Bettati S. Pucci A.M. Burkhard P. Cook P.F. J. Mol. Biol. 1998; 283: 135-146Crossref PubMed Scopus (20) Google Scholar), several catalytic intermediates were isolated and characterized in the crystalline state, opening the way to their structural determination. We have recently expressed and purified to near homogeneity recombinant human CBS comprising amino acid residues 2–413. This enzyme, missing 138 C-terminal residues, forms dimers, is not activated by S-adenosyl-l-methionine, and does not exhibit the aggregating properties of the full-length enzyme. In addition, the recombinant CBS polypeptide contains a 23-amino acid spacer at its N terminus. The truncated enzyme still binds PLP and heme and is about 2 times more active than the full-length CBS (25Janosik, M., Meier, M., Kery, V., Oliveriusova, J., Burkhard, P, and Kraus, J. P. (2000) Acta Crystallogr., in pressGoogle Scholar). Crystals of the recombinant active core of CBS have been obtained, and the three-dimensional structure is being determined (26McPherson A. Preparation and Analysis of Protein Crystals. John Wiley & Sons, New York1982: 94-96Google Scholar). Here, we have studied the reactivity of these crystals by polarized absorption microspectrophotometry as an essential prerequisite to the crystallographic analysis of the enzyme and the structure to function correlation.CONCLUSIONCryo-crystallography (33Douzo P. Petsko G.A. Adv. Protein Chem. 1984; 36: 245-361Crossref PubMed Scopus (101) Google Scholar) and kinetic crystallography (34Moffat K. Annu. Rev. Biophys. Chem. 1989; 18: 309-332Crossref PubMed Scopus (123) Google Scholar), making use of either slowly reacting substrates and substrate analogues (20Rossi G.L. Bernhard S.A. J. Mol. Biol. 1970; 49: 85-91Crossref PubMed Scopus (37) Google Scholar) or slowly reacting mutant enzymes (35Bolduc J.L. Dyer D.H. Scott W.G. Singer P. Sweet R.M. Koshland D.E. Stoddard B.L. Science. 1995; 268: 1312-1318Crossref PubMed Scopus (112) Google Scholar), have considerably expanded the capability to detect and characterize not only the native form of enzymes and proteins but also transiently accumulating species. Detailed functional studies of protein crystals by spectroscopic techniques have allowed us to define the experimental conditions for the accumulation of catalytic intermediates, thus directing the crystallographic measurements. These conditions are not always similar to those derived by experiments in solution, because crystal lattice forces affect the relative stability of catalytic intermediates in unpredictable ways. Examples are the different effect of cations on the accumulation of the quinonoid species of tryptophan synthase in the crystal and in solution (22Mozzarelli A. Peracchi A. Rovegno B. Dale G. Rossi G.L. Dunn M.F. J. Biol. Chem. 2000; 275: 6956-6962Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) and the different affinity of the natural substrate O-acetylserine to several crystal forms of O-acetylserine sulfhydrylase, where one form is 500-fold less active than the enzyme in solution and another is completely inactive (24Mozzarelli A. Bettati S. Pucci A.M. Burkhard P. Cook P.F. J. Mol. Biol. 1998; 283: 135-146Crossref PubMed Scopus (20) Google Scholar).The present investigation of the active core of human CBS crystals has allowed to prepare the oxidized and reduced forms of the enzyme, the heme-free protein, and the key catalytic intermediate α-aminoacrylate. For the first time, it has also been demonstrated that the heme does not participate in PLP-dependent catalysis of CBS. However, further investigations in solution are required to assess the fine-tuning of ligand binding and catalysis by the heme moiety. High plasmatic levels of homocysteine have recently been associated with an increased risk of cardiovascular disease (1Refsum H. Ueland P. Nygard O. Vollset S.E. Annu. Rev. Med. 1998; 49: 31-62Crossref PubMed Scopus (1832) Google Scholar). Homocysteine is formed from S-adenosylhomocysteine and is either removed by cystathionine β-synthase (EC 4.2.1.22, CBS)1 in the trans-sulfuration pathway or remethylated to methionine in the methionine cycle. Deficiency of CBS is the major cause of inherited homocystinuria. CBS is a pyridoxal 5′-phosphate (PLP)-dependent enzyme catalyzing the synthesis of cystathionine from homocysteine and l-serine. The reaction proceeds via a β-replacement mechanism, similar to that of tryptophan synthase and O-acetylserine sulfhydrylase (2Borcsok E. Abeles R.H. Arch. Biochem. Biophys. 1982; 213: 695-707Crossref PubMed Scopus (54) Google Scholar). These enzymes belong to the β-family and fold II type within the PLP-dependent enzymes classification (3Alexander F.W. Sandmeier E. Metha P.K. Christen P. Eur. J. Biochem. 1994; 291: 953-960Crossref Scopus (343) Google Scholar, 4Grishin N.V. Phillips M.A. Goldsmith E.J. Protein Sci. 1995; 4: 1291-1304Crossref PubMed Scopus (339) Google Scholar). Other members of the β-family are serine and threonine dehydratases. The human CBS is a 63-kDa homotetramer containing one PLP and one heme per subunit (5Kery V. Bukovska G. Kraus J.P. J. Biol. Chem. 1994; 269: 25283-25288Abstract Full Text PDF PubMed Google Scholar). Whereas the functional role of PLP is known, the role of the heme is less clear. It was demonstrated that the heme redox state affects the affinity of the enzyme for the substrates (6Taoka S. Ohja S. Shan X.Y. Kruger W.D. Banerjee R. J. Biol. Chem. 1998; 273: 25179-25184Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Moreover, the catalytic activity of CBS is controlled by S-adenosylmethionine, which specifically binds to a C-terminal site. The trypsinolysis of a 18 kDa C-terminal fragment leads to a dimeric form, 2-fold more active than the native species and no longer regulated by S-adenosylmethionine (7Kery V. Poneleit L. Kraus J.P. Arch. Biochem. Biophys. 1998; 355: 222-232Crossref PubMed Scopus (138) Google Scholar). Similar results have been obtained on other truncated forms of CBS, obtained by insertion of nonsense mutations (8Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38: 13155-13161Crossref PubMed Scopus (82) Google Scholar). Investigation of the catalytic reaction brought about by PLP is complicated by the overlapping chromophoric properties of the heme. The recent investigation of the yeast enzyme, which does not contain heme groups, permitted a better characterization of the PLP role in the catalytic steps (9Jhee K.H. McPhie P. Miles E.W. J. Biol. Chem. 2000; 275: 11541-11544Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 10Jhee K.H. McPhie P. Miles E.W. Biochemistry. 2000; 39: 10548-10556Crossref PubMed Scopus (81) Google Scholar). An α-aminoacrylate species, absorbing at 470 and 320 nm, was observed upon reaction with l-serine. The nucleophilic attack of homocysteine on the α-aminoacrylate led to the formation of the product cystathionine. However, it is not yet known how closely the functional properties of the yeast enzyme resemble those of the human source. Structural studies of catalytic intermediates of tryptophan synthase and O-acetylserine sulfhydrylase (11Miles E.W. Adv. Enzymol. Relat. Areas Mol. Biol. 1991; 64: 93-172PubMed Google Scholar, 12Cook P.F. Hara S. Nalabolu S. Schnackerz K.D. Biochemistry. 1992; 31: 2298-2303Crossref PubMed Scopus (62) Google Scholar, 13Hyde C.C. Ahmed S.A. Padlan E.A. Miles E.W. Davies D.R. J. Biol. Chem. 1988; 263: 17857-17871Abstract Full Text PDF PubMed Google Scholar, 14Burkhard P. Rao G.S. Hohenester E. Schnackerz K.D. Cook P.F. Jansonius J.N. J. Mol. Biol. 1998; 283: 121-133Crossref PubMed Scopus (179) Google Scholar, 15Schneider T.R. Gerhardt E. Lee M. Liang P.H. Anderson K.S. Schlichting I. Biochemistry. 1998; 37: 5394-5406Crossref PubMed Scopus (134) Google Scholar, 16Rhee S. Parris K.D. Hyde C.C. Ahmed S.A. Miles E.W. Davies D.R. Biochemistry. 1997; 36: 7664-7680Crossref PubMed Scopus (143) Google Scholar, 17Rhee S. Miles E.W. Mozzarelli A. Davies D.R. Biochemistry. 1998; 37: 10653-10659Crossref PubMed Scopus (40) Google Scholar, 18Burkhard P. Tai C.H. Ristroph C.M. Cook P.F. Jansonius J.N. J. Mol. Biol. 1999; 291: 941-953Crossref PubMed Scopus (119) Google Scholar) have unveiled the nature of enzyme action. A common feature is an open to closed conformational transition of the active site taking place along the catalytic pathway. To fully exploit the structural information as well as to determine the structure of as many as possible catalytic intermediates, it is of paramount relevance to investigate the functional properties of the enzyme in the crystalline state by polarized absorption microspectrophotometry (19Mozzarelli A. Rossi G.L. Annu. Rev. Biophys. Biomol. Struct. 1996; 25: 343-365Crossref PubMed Scopus (104) Google Scholar). This approach was pioneered in the late sixties by Rossi and Bernhard (20Rossi G.L. Bernhard S.A. J. Mol. Biol. 1970; 49: 85-91Crossref PubMed Scopus (37) Google Scholar). In the case of tryptophan synthase (17Rhee S. Miles E.W. Mozzarelli A. Davies D.R. Biochemistry. 1998; 37: 10653-10659Crossref PubMed Scopus (40) Google Scholar, 21Mozzarelli A. Peracchi A. Rossi G.L. Ahmed S.A. Miles E.W. J. Biol. Chem. 1989; 264: 15774-15780Abstract Full Text PDF PubMed Google Scholar, 22Mozzarelli A. Peracchi A. Rovegno B. Dale G. Rossi G.L. Dunn M.F. J. Biol. Chem. 2000; 275: 6956-6962Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 23Peracchi A. Mozzarelli A. Rossi G.L. Biochemistry. 1995; 34: 9459-9465Crossref PubMed Scopus (78) Google Scholar) and O-acetylserine sulfhydrylase (24Mozzarelli A. Bettati S. Pucci A.M. Burkhard P. Cook P.F. J. Mol. Biol. 1998; 283: 135-146Crossref PubMed Scopus (20) Google Scholar), several catalytic intermediates were isolated and characterized in the crystalline state, opening the way to their structural determination. We have recently expressed and purified to near homogeneity recombinant human CBS comprising amino acid residues 2–413. This enzyme, missing 138 C-terminal residues, forms dimers, is not activated by S-adenosyl-l-methionine, and does not exhibit the aggregating properties of the full-length enzyme. In addition, the recombinant CBS polypeptide contains a 23-amino acid spacer at its N terminus. The truncated enzyme still binds PLP and heme and is about 2 times more active than the full-length CBS (25Janosik, M., Meier, M., Kery, V., Oliveriusova, J., Burkhard, P, and Kraus, J. P. (2000) Acta Crystallogr., in pressGoogle Scholar). Crystals of the recombinant active core of CBS have been obtained, and the three-dimensional structure is being determined (26McPherson A. Preparation and Analysis of Protein Crystals. John Wiley & Sons, New York1982: 94-96Google Scholar). Here, we have studied the reactivity of these crystals by polarized absorption microspectrophotometry as an essential prerequisite to the crystallographic analysis of the enzyme and the structure to function correlation. CONCLUSIONCryo-crystallography (33Douzo P. Petsko G.A. Adv. Protein Chem. 1984; 36: 245-361Crossref PubMed Scopus (101) Google Scholar) and kinetic crystallography (34Moffat K. Annu. Rev. Biophys. Chem. 1989; 18: 309-332Crossref PubMed Scopus (123) Google Scholar), making use of either slowly reacting substrates and substrate analogues (20Rossi G.L. Bernhard S.A. J. Mol. Biol. 1970; 49: 85-91Crossref PubMed Scopus (37) Google Scholar) or slowly reacting mutant enzymes (35Bolduc J.L. Dyer D.H. Scott W.G. Singer P. Sweet R.M. Koshland D.E. Stoddard B.L. Science. 1995; 268: 1312-1318Crossref PubMed Scopus (112) Google Scholar), have considerably expanded the capability to detect and characterize not only the native form of enzymes and proteins but also transiently accumulating species. Detailed functional studies of protein crystals by spectroscopic techniques have allowed us to define the experimental conditions for the accumulation of catalytic intermediates, thus directing the crystallographic measurements. These conditions are not always similar to those derived by experiments in solution, because crystal lattice forces affect the relative stability of catalytic intermediates in unpredictable ways. Examples are the different effect of cations on the accumulation of the quinonoid species of tryptophan synthase in the crystal and in solution (22Mozzarelli A. Peracchi A. Rovegno B. Dale G. Rossi G.L. Dunn M.F. J. Biol. Chem. 2000; 275: 6956-6962Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) and the different affinity of the natural substrate O-acetylserine to several crystal forms of O-acetylserine sulfhydrylase, where one form is 500-fold less active than the enzyme in solution and another is completely inactive (24Mozzarelli A. Bettati S. Pucci A.M. Burkhard P. Cook P.F. J. Mol. Biol. 1998; 283: 135-146Crossref PubMed Scopus (20) Google Scholar).The present investigation of the active core of human CBS crystals has allowed to prepare the oxidized and reduced forms of the enzyme, the heme-free protein, and the key catalytic intermediate α-aminoacrylate. For the first time, it has also been demonstrated that the heme does not participate in PLP-dependent catalysis of CBS. However, further investigations in solution are required to assess the fine-tuning of ligand binding and catalysis by the heme moiety. Cryo-crystallography (33Douzo P. Petsko G.A. Adv. Protein Chem. 1984; 36: 245-361Crossref PubMed Scopus (101) Google Scholar) and kinetic crystallography (34Moffat K. Annu. Rev. Biophys. Chem. 1989; 18: 309-332Crossref PubMed Scopus (123) Google Scholar), making use of either slowly reacting substrates and substrate analogues (20Rossi G.L. Bernhard S.A. J. Mol. Biol. 1970; 49: 85-91Crossref PubMed Scopus (37) Google Scholar) or slowly reacting mutant enzymes (35Bolduc J.L. Dyer D.H. Scott W.G. Singer P. Sweet R.M. Koshland D.E. Stoddard B.L. Science. 1995; 268: 1312-1318Crossref PubMed Scopus (112) Google Scholar), have considerably expanded the capability to detect and characterize not only the native form of enzymes and proteins but also transiently accumulating species. Detailed functional studies of protein crystals by spectroscopic techniques have allowed us to define the experimental conditions for the accumulation of catalytic intermediates, thus directing the crystallographic measurements. These conditions are not always similar to those derived by experiments in solution, because crystal lattice forces affect the relative stability of catalytic intermediates in unpredictable ways. Examples are the different effect of cations on the accumulation of the quinonoid species of tryptophan synthase in the crystal and in solution (22Mozzarelli A. Peracchi A. Rovegno B. Dale G. Rossi G.L. Dunn M.F. J. Biol. Chem. 2000; 275: 6956-6962Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) and the different affinity of the natural substrate O-acetylserine to several crystal forms of O-acetylserine sulfhydrylase, where one form is 500-fold less active than the enzyme in solution and another is completely inactive (24Mozzarelli A. Bettati S. Pucci A.M. Burkhard P. Cook P.F. J. Mol. Biol. 1998; 283: 135-146Crossref PubMed Scopus (20) Google Scholar)." @default.
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• W2000537633 title "Functional Properties of the Active Core of Human Cystathionine β-Synthase Crystals" @default.
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