![]() Koroleva OV, Stepanova EV, Binukov VI, Timofeev VP, Pfeil W (2001) Biochim Biophys Acta 1547:397–407įersht A (1999) Structure and mechanism in protein science. ![]() ![]() Kelly SM, Price NC (1997) Biochim Biophys Acta 1338:161–185īonomo RP, Cennamo G, Purrello R, Santoro AM, Zappala RJ (2001) Inorg Chem 83:67–75Īgostinelli E, Cervoni L, Giartosio A, Morpurgo L (1995) Biochem J 306:697–702 Lakowicz JR (1999) Principles of fluorescence spectroscopy. Moser CC, Dutton PL (1996) In: Bendall DS (ed) Protein electron transfer. Kataoka K, Kitagawa R, Inoue M, Naruse D, Sakurai T, Huang H-W (2005) Biochemistry 44:7004–7012 Hall JF, Kanbi LD, Strange RW, Hasnain SS (1999) Biochemistry 38:12675–12680ĭiederix REM, Canters GW, Dennison C (2000) Biochemistry 39:9551–9560 Pascher T, Karlsson BG, Nordling M, Malmström BG, Vänngård T (1993) Eur J Biochem 212:289–296 Palmer AE, Randall DW, Xu F, Solomon EI (1999) J Am Chem Soc 121:7138–7149 Xu F, Palmer AE, Yaver DS, Berka RM, Gambeta GA, Brown SH, Solomon EI (1999) J Biol Chem 274:12372–12375 Xu F, Berka RM, Wahleithner JA, Nelson BA, Shuster JR, Brown SH, Palmer AE, Solomon EI (1998) Biochem J 334:63–70 Garavaglia S, Cambria MT, Miglio M, Ragusa S, Iacobazzi V, Palmieri F, D’Ambrosio C, Scaloni A, Rizzi M (2004) J Mol Biol 342:1519–1531ĭeLano WL (2002) The PyMOL molecular graphics system. Hakulinen N, Kiiskinen LL, Kruus K, Saloheimo M, Paananen A, Koivula A, Rouvinen J (2002) Nat Struct Biol 9:601–605 Ramachandran GN, Sasisekharan V (1968) Adv Protein Chem 23:283–437ĭucros V, Brzozowski AM, Wilson KS, Ostergaard P, Schneider P, Svendson A, Davies GJ (2001) Acta Crystallogr Sect D 57:333–336 Monsellier E, Bedouelle H (2005) Protein Eng Des Sel 18:445–456īrenner AJ, Harris ED (1995) Anal Biochem 226:80–84 Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) J Appl Crystallogr 26:283–291 Ramakrishnan C, Ramachandran GN (1965) Biophys J 5:909–933 Murshudov GN, Vagin AA, Lebedev A, Wilson KS, Dodson EJ (1999) Acta Crystallogr Sect D 55:247–255Įmsley P, Cowtan K (2004) Acta Crystallogr Sect D 60:2126–2132 Otwinowski Z, Minor W (1997) In: Carter CW Jr, Sweet RM (eds) Methods in enzymology, macromolecular crystallography, vol 276. Karlin S, Zhu SZY, Karlin KD (1997) Proc Natl Acad Sci USA 94:14225–14230 Solomon EI, Sundaram UM, Machonkin TE (1996) Chem Rev 96:2563–2605 Martins LO, Soares CM, Pereira MM, Teixeira M, Costa T, Jones GH, Henriques AO (2002) J Biol Chem 277:18849–18859Įnguita FJ, Martins LO, Henriques AO, Carrondo MA (2003) J Biol Chem 278:19416–19425Įnguita FJ, Marçal D, Martins LO, Grenha R, Henriques AO, Lindley PF, Carrondo MA (2004) J Biol Chem 279:23472–23476īento I, Martins LO, Lopes GG, Arménia MA, Lindley PF (2005) Dalton Trans 21:3507–3513 Gianfreda L, Xu F, Bollag J-M (1999) Bioremediation J 3:1–25 Xu F (1999) In: Flickinger MC, Drewn SW (eds) Encyclopedia of bioprocess technology: fermentation, biocatalysis and bioseparation. Lindley PF (2001) In: Bertini I, Sigel A, Sigel H (eds) Multi-copper oxidases. Messerschmidt A (1997) Multi-copper oxidases. ![]() The T1 copper centre clearly plays a key role, from the structural, catalytic and stability viewpoints, in the regulation of CotA laccase activity. At 1.9 M guanidinium hydrochloride, half of the molecules are in an intermediate conformation, only slightly less stable than the native state (approximately 1.4 kcal/mol). Whilst the unfolding of the tertiary structure in the wild-type enzyme is a two-state process displaying a midpoint at a guanidinium hydrochloride concentration of 4.6 M and a free-energy exchange in water of 10 kcal/mol, the unfolding for both mutant enzymes is clearly not a two-state process. ![]() T1 copper depletion is a key event in the inactivation and thus it is a determinant of the thermodynamic stability of wild-type and mutant proteins. However the M502L mutant exhibits a twofold to fourfold decrease in the k cat values for the all substrates tested and the catalytic activity in M502F is even more severely compromised 10% activity and 0.15–0.05% for the non-phenolic substrates and for the phenolic substrates tested when compared with the wild-type enzyme. The replacement of the weak so-called axial ligand of the T1 site leads to an increase in the redox potential by approximately 100 mV relative to that of the wild-type enzyme ( E 0=455 mV). X-ray structural comparison of M502L and M502F mutants with the wild-type CotA shows that the geometry of the T1 copper site is maintained as well as the overall fold of the proteins. Site-directed mutagenesis has been used to replace Met502 in CotA laccase by the residues leucine and phenylalanine. ![]()
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