molecular and active-site structure of tyrosinase

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02-UTILIZATION & PROCESSING 311 LOO9 MOLECULARAND ACTIVE-SITE SrRUcrURE OF TYPGSINASE. M.P. Jackman, M. Huber a&K. Perch. Biochemisches Institut der Universitgt Ziirich, Winterthurerstrasse 196, CH-8057 Ziirich, Switzerland. Tyrosinase is a copper-containing monooxygenase catalyzing both the o-hydroxylation of mmphenols and aromatic aminesand the oxida- tion-ofo-diphenols to o-quinones and o-aminophenols to o-quinoneimines. The acti'iTe site of the- enzymeconsists of a coupledbynuclear copper centerwhich interacts with both molecular oxygenand organicsubstra- tes. Spectroscopic ard protein sequencing data have shownthat tyrosi- nase is structurally relatedto the oxygen-transporting hemocyanins. To elucidate the factorsinfluencing oxygenand substrate bindingin tyro- sinase, we have performed site-directedmutagenesis of the gene from the bacterium Streptomycesglaucescens. Chemical and spectroscopic properties of the mutantenzymeswill be presented. L(-)l() BINDING AND AC’?IVATION OF DIOXYGEN BY COPPERCOMPLEXES. A. D. Zubsrbiihler, University of Basel,Basel,Switzerland. Bindingand Activation of dioxygen by coppercomplexes has long been based purelyon kinetic evidence for low molecular systems. With the developarent and characterization (1) of quasireversible p-peroxo coppercomplexes it has beccrme possible to obtainthe thermodynamic and kineticparameters of dioxygen bindingand oxygenation of organic substrates. Enthalpies of formation of such adducts are closeto those of iron complexes and of biological dioxygen carriers, i.e. roughly 60 kJ/mol,reaction entropies being responsible for theirlow stability constants at room temperature. Experimental evidence is indicating a strongelectrophile as the oxygenating species. Dioxygen activation occursboth thermally and photochemically. [ll K.D. Kariinet-al.,J. Am. &em. Sot.,106, 6769 (1988). Loll Robert SPECTROSCOPIC AND SITE-DIRECTED MUTAGENESIS STUDIES ON Cu2Zn2SOD. Lucia Banci, Ivano Bertini, Claudio Luchinat, and A. Hallewell - University of Florence, Florence, University of 3ologna Italy and Chiron Corporation, Emeryville, CA, U.S.A. Copper-zinc superoxide dismutases catalyze in vitro the reaction of dismutation of superoxide anion and seem to have, also in vivo, a role of protection against oxygen toxicity. The catalytic mechanism for Cu2Zn2SOD is still an open i fascinating problem. Spectroscopic studies, particularly through H NMRD measurements, 'H NMR spectra on the fully active Cu2CO2 derivative, and site directed mutagenesis on some aminoacid residues present in the active cavity that are thought to have a key role in the enzymatic reaction (Arg-143, Thr-137, Glu-133, Glu-132, ~~~-124, Asp-1251, have shed light on the reaction mechanism. Aniosn bind copper and some of them act as inhibitors. They induce changes in the coordination polyhedron which are discussed in detail.

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02-UTILIZATION & PROCESSING 311

LOO9 MOLECULARAND ACTIVE-SITE SrRUcrURE OF TYPGSINASE. M.P. Jackman, M. Huber a&K. Perch. Biochemisches Institut der Universitgt

Ziirich, Winterthurerstrasse 196, CH-8057 Ziirich, Switzerland. Tyrosinase is a copper-containing monooxygenase catalyzing both

the o-hydroxylation of mmphenols and aromatic amines and the oxida- tion-of o-diphenols to o-quinones and o-aminophenols to o-quinoneimines. The acti'iTe site of the- enzyme consists of a coupled bynuclear copper center which interacts with both molecular oxygen and organic substra- tes. Spectroscopic ard protein sequencing data have shown that tyrosi- nase is structurally related to the oxygen-transporting hemocyanins. To elucidate the factors influencing oxygen and substrate binding in tyro- sinase, we have performed site-directed mutagenesis of the gene from the bacterium Streptomyces glaucescens. Chemical and spectroscopic properties of the mutant enzymes will be presented.

L(-)l() BINDING AND AC’?IVATION OF DIOXYGEN BY COPPER COMPLEXES. A. D. Zubsrbiihler, University of Basel, Basel, Switzerland. Binding and Activation of dioxygen by copper complexes has long

been based purely on kinetic evidence for low molecular systems. With the developarent and characterization (1) of quasireversible p-peroxo copper complexes it has beccrme possible to obtain the thermodynamic and kinetic parameters of dioxygen binding and oxygenation of organic substrates. Enthalpies of formation of such adducts are close to those of iron complexes and of biological dioxygen carriers, i.e. roughly 60 kJ/mol, reaction entropies being responsible for their low stability constants at room temperature. Experimental evidence is indicating a strong electrophile as the oxygenating species. Dioxygen activation occurs both thermally and photochemically.

[ll K.D. Kariin et-al., J. Am. &em. Sot., 106, 6769 (1988).

Loll Robert

SPECTROSCOPIC AND SITE-DIRECTED MUTAGENESIS STUDIES ON Cu2Zn2SOD. Lucia Banci, Ivano Bertini, Claudio Luchinat, and A. Hallewell - University of Florence, Florence, University of

3ologna Italy and Chiron Corporation, Emeryville, CA, U.S.A. Copper-zinc superoxide dismutases catalyze in vitro the reaction

of dismutation of superoxide anion and seem to have, also in vivo, a role of protection against oxygen toxicity. The catalytic mechanism for Cu2Zn2SOD is still an open

i fascinating problem. Spectroscopic

studies, particularly through H NMRD measurements, 'H NMR spectra on the fully active Cu2CO2 derivative, and site directed mutagenesis on some aminoacid residues present in the active cavity that are thought to have a key role in the enzymatic reaction (Arg-143, Thr-137, Glu-133, Glu-132, ~~~-124, Asp-1251, have shed light on the reaction mechanism. Aniosn bind copper and some of them act as inhibitors. They induce changes in the coordination polyhedron which are discussed in detail.