1. Fig 4: Energetically favoured poses of ccordination for tested substrates Tyrosinase (Ty) is a type III copper containing protein which catalyzes the hydroxylation of monophenols to ortho-diphenols and the subsequent two electron oxidation to ortho-quinones with molecular oxygen (Scheme 1) (7). After the publication of the crystallographic structure of Streptomyces Castaneoglobisporus Ty (1) it has been demonstrated that also oxy-Ty binds dioxygen as peroxide in the unique side-on bridging ( μ-η 2 : η 2 ) manner. Some details of mechanism of reaction remains however unclear, with different hypothesis that still wait for direct experimental prove. Among these the DFT theoretical detailed description proposed by Siegbahn PE (2) in which a novel oxygen coordination is supposed, with a hydroperoxide structure for the reaction centre (Fig 2 a). This structure has not been proved spectroscopically and can be hardly reconciled with the crystallographic structure, where the structure of the peroxo is that shown in Fig1. Another recent hypothesis of mechanism, solely based on the observation of the recently released crystal structure, has been proposed by Decker et al (3), which implies coordination to CuA (the copper are classified as CuA coordinated with H38, H54, H63 and CuB coordinated to H190, H194, H216) and a reorientation of the peroxo bridge before reaction with the aromatic ring (see Fig 2 b). In this study a combined experimental and computations approach to the molecular mechanism of Ty reactivity (based on the S. antibiboticus) toward substituted monophenols (three isomers of mono-cholorphenols (MCP), 2-CP, 3-CP, 4-CP) and naphtols (1-naphtol and 2- naphtol) are presented Fig 2: a) reactive structure of oxy-Ty for the scheme of reaction proposed by DFT analysis (Siegbahn PE, 2004). This structure has not been observed experimentally. b) rearrangement of the peroxo bridge after substrate coordination (to CuA) according to the recent hypothesis by Decker and co-workers (Decker et al, 2005); the base proposed is H194, with a intially deprotonated at the N-δ. However, paramagnetic MMR on the met –Ty indicate a protonated state for all coordinated histidine nitogens (4). Fig 1: Scheme of monophenolasic reaction Fig1... b) a) b) “Mechanical investigation into S.antibioticus Tyrosinase acivity toward chlorophenols and naphtols” Stefano Marino a, Stefano Fogal a, Stefano Moro b Luca De Gioia c, Luigi Casella d and Luigi Bubacco a a Department of Biology, b Molecular Modeling Section, Department of Pharmacology University of Padova, Italy. c Department of Biotechnology and Biosciences, University of Milano “Bicocca”, Italy d Department of Chemistry, University of Pavia, Italy d) b) Conclusions: Phenolic substrate should be de-protonated before coordination to CuB. The potential proton acceptors are either the molecular oxygen thought proton transfer from O1 to O2 or one of purported proton shuttle (E182, N191 that are above bound O 2 in the structure) After coordination, O1 seems to be the best candidate for reaction to the aromatic ring, in the nearest orto- position In the docking analysis upon binding of all investigated MCPs, the alogen atoms are always found at the larger distance possible from the binuclear centre hydroxylation. This implies a stereospecifricity (to position C6) that is a known characteristic of this enzyme. Consequentially, for 2CP sterical effects of chloride atom hider a useful coordination since it imposes for the C6 atom a position that is too far from O1 (about 5 A) 2CP remain a good competitive inhibitor inhibits since all energy parameters explored in the docking favour the access to the enzymatic cleft For 1-naphtol the values of the energy parameters explored suggests a weak tendency to approach to the reaction centre (in the phenolic form). The few bound molecules that approach the site gives an “unproductive” coordination pose (with the C10 near to the molecular oxygen O1) that rationalize the lack of reactivity and undetectable inhibition. References: 1) Matoba Y, Kumagai T, Yamamoto A, Yoshitsu H, Sugiyama M. (2006) J Biol Chem, 28(13): 8981-90 2) Siegbahn PE, (2004) J Biol Inorg Chem., 9(5):577-90 3) Decker H, Schweikardt T, Tuczek F (2006) Angew Chem Int Ed Engl,45(28):4546-50 4) Bubacco L, Salgado J, Tepper AW, Vijgenboom E, Canters GW (1999) FEBS Lett, 442(2-3):215-20. 5) Baxter, C.A., Murray, C.W., Clark, D.E., Westhead, D.R. and Eldridge, M.D. (1998) Proteins: Structure, Function and Genetics 33:367. 6) Halgren, T.A. J. (1996) Comput. Chem, 17:490 7) van Gastel M, Bubacco L, Groenen EJ, Vijgenboom E, Canters GW. (2000) FEBS Lett,474(2-3): 228-32. 3) Mechanical investigation of the precoordination poses for the 3) Mechanical investigation of the precoordination poses for the deprotonated substrates deprotonated substrates Strategy: starting from docking poses coordinates, substrates were de- protonated and then the potential surface was sampled and complex structures were minimized with AMBER99. >> Results shown in Fig 4: a] 1-naphtol: naphtolate oxygen near to CuB, with Hydroxilable carbon (C2) in a not reactive position (opposite to RC) b] 2-naptol: pose is consistent with reactivity, with naphtolate oxygen near to CuB (2 A) and orto-hydroxylable carbon (C1) near to one of the coordinated oxygens (O1 at 2.2 A) c] 4CP: best pose similar to that of 2-naphtol (trans to H190), while best phenolic pose was trans toH216) : approach to CuB (1.8 A) and C2/C6 near to O1 (2.2 A) - 3CP (not shown in Fig 4): best pose very similar to that of 4CP with C6 position in a reactive position near to O1(fig 6) d] 2CP best coordination pose: - lateral position of the pheonolic ring (near to H194) - higher distance to CuB (about 2.3 A) - orto-hydroxylable C6 too far from coordinated oxygens (4.9 A from O1) Results: 1) Kinetic measurments 1) Kinetic measurments (Ty 0.1 μM, in 100 mM phosphate buffer pH 6.8 ) 4-CP : K m =0.973 mM 2-naphtol: K m = 0.75 mM 3-CP : K m = 28.57 mM 1-napthol: not reactive 2-CP: K i = 7.5mM 2-CP (inhibiton kinetcis with L-DOPA as substrate) resulted to be a competitive inhibitor, while 1-naphtol revealed to be neither a substrate or an inhibitor. 2) Docking to the reaction centre (RC) for protonated substrates - The presented structure was obtained starting from the structure 1wx2 (Streptomyces Castaneoglobisporus oxy-Ty) by Homology Modelling with MOE2006 (82% identity with S. antibioticus Ty ) - RC treatment with Spartan02 (with protonated His N-δ), docking with flexible MOE-Dock methodology (GA protocol, MMFF94x)(5,6) > Results for docking of mono-chloro-phenols are shown in Fig2: three isomers have a similar approach to the RC but with different enegetic scores (-60 kcal/mol for 4CP, -50 kcal/mol for 2CP, -2 kcal/mol for 3CP) > Results for docking of napthols are shown in Fig3: a different approach to RC characterize the isomers, with 2-naphtol pointing to O1( Energy = -7 kcal/mol) and 1-naphtol pointing to O2 with a lower energy score of - 2 kcal/mol Fig1... O1 (q = - 0.35) O2 (q = - 0.27) a) c) Fig2: Best docking poses for 2CP (yellow), 4CP (orange). 3Cp, not shown (E = -2 kcal/mol) has a pose highly superimposable to that of 4Cp. Coordinated oxygens resulted to be asymmetrically coordinated with a different calculated fractional charges: O1 (as enumerated in 1wx2) charge is -0.35 while O2 characterizing value is -0.27 Fig3: Best docking poses for 1-naphtol (yellow) and 2-naphtol (green) Fig6: Hypothetical scheme of reaction. To enhance the stereochemical selectivity operated by the protein before coordination, a meta substituted generic phenol is shown. A) In our analysis approach to metallic centre occurs with alogen pointing out of reaction centre. In the first part of the reaction the overidding interaction is π-π stacking interaction between H194 and susbtrate's phenolic ring. B) Subsequently an H-bonding interaction may occur between oxydrilic Oxygen and coordinated O1, and this intearaction becomes predominant on the stacking one, with susbtrates that re-orientate in space as depicted in figure. C) After deprotonation phenolate coordinates to CuB. The proton may be shifted away from reaction centre by a generic base D) After coordination and proton transfer the peroxo bridge is broken, triggering the second part of the reaction, in which O1 attack (or be attacked from) the phenolic ring in C6 position and O2 exits reaction centre as a water molecule. A)B) C) D)