- Email: [email protected]
Molecular and active-site structure of tyrosinase
02-UTILIZATION
& PROCESSING
311
MOLECULARAND ACTIVE-SITESrRUcrUREOF TYPGSINASE.M.P. Jackman, LOO9 M. Huber a&K. Perch. BiochemischesInstitutder Universitgt Ziirich, Winterthurerstrasse 196, CH-8057 Ziirich, Switzerland. Tyrosinaseis a copper-containing monooxygenase catalyzingboth the o-hydroxylation of mmphenols and aromaticaminesand the oxidation-ofo-diphenolsto o-quinonesand o-aminophenols to o-quinoneimines. The acti'iTe site of the- enzyme consists of a coupledbynuclearcopper centerwhich interactswith both molecularoxygenand organicsubstrates. Spectroscopic ard protein sequencingdata have shown that tyrosinase is structurallyrelatedto the oxygen-transporting hemocyanins.To elucidatethe factorsinfluencingoxygen and substratebinding in tyrosinase,we have performed site-directedmutagenesisof the gene from the bacterium Streptomyces glaucescens.Chemical and spectroscopic propertiesof the mutant enzymeswill be presented.
L(-)l()
BINDING AND AC’?IVATION OF DIOXYGENBY COPPERCOMPLEXES. A. D. Zubsrbiihler, Universityof Basel,Basel, Switzerland. Bindingand Activationof dioxygenby coppercomplexeshas long been based purely on kineticevidencefor low molecularsystems.With the developarent and characterization (1) of quasireversible p-peroxo copper complexesit has beccrme possibleto obtain the thermodynamic and kineticparametersof dioxygenbindingand oxygenationof organic substrates.Enthalpiesof formationof such adductsare close to those of iron complexesand of biologicaldioxygencarriers,i.e. roughly60 kJ/mol, reactionentropiesbeing responsiblefor their low stability constantsat room temperature. Experimental evidenceis indicatinga strongelectrophileas the oxygenatingspecies.Dioxygenactivation occurs both thermallyand photochemically. [ll K.D. Kariinet-al.,J. Am. &em. Sot.,106, 6769 (1988).
Loll
SPECTROSCOPIC AND SITE-DIRECTED MUTAGENESIS STUDIES ON Cu2Zn2SOD. Lucia Banci, Ivano Bertini, Claudio Luchinat, and Robert 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 fascinating problem. Spectroscopic studies, particularly through iH 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.
& PROCESSING
311
MOLECULARAND ACTIVE-SITESrRUcrUREOF TYPGSINASE.M.P. Jackman, LOO9 M. Huber a&K. Perch. BiochemischesInstitutder Universitgt Ziirich, Winterthurerstrasse 196, CH-8057 Ziirich, Switzerland. Tyrosinaseis a copper-containing monooxygenase catalyzingboth the o-hydroxylation of mmphenols and aromaticaminesand the oxidation-ofo-diphenolsto o-quinonesand o-aminophenols to o-quinoneimines. The acti'iTe site of the- enzyme consists of a coupledbynuclearcopper centerwhich interactswith both molecularoxygenand organicsubstrates. Spectroscopic ard protein sequencingdata have shown that tyrosinase is structurallyrelatedto the oxygen-transporting hemocyanins.To elucidatethe factorsinfluencingoxygen and substratebinding in tyrosinase,we have performed site-directedmutagenesisof the gene from the bacterium Streptomyces glaucescens.Chemical and spectroscopic propertiesof the mutant enzymeswill be presented.
L(-)l()
BINDING AND AC’?IVATION OF DIOXYGENBY COPPERCOMPLEXES. A. D. Zubsrbiihler, Universityof Basel,Basel, Switzerland. Bindingand Activationof dioxygenby coppercomplexeshas long been based purely on kineticevidencefor low molecularsystems.With the developarent and characterization (1) of quasireversible p-peroxo copper complexesit has beccrme possibleto obtain the thermodynamic and kineticparametersof dioxygenbindingand oxygenationof organic substrates.Enthalpiesof formationof such adductsare close to those of iron complexesand of biologicaldioxygencarriers,i.e. roughly60 kJ/mol, reactionentropiesbeing responsiblefor their low stability constantsat room temperature. Experimental evidenceis indicatinga strongelectrophileas the oxygenatingspecies.Dioxygenactivation occurs both thermallyand photochemically. [ll K.D. Kariinet-al.,J. Am. &em. Sot.,106, 6769 (1988).
Loll
SPECTROSCOPIC AND SITE-DIRECTED MUTAGENESIS STUDIES ON Cu2Zn2SOD. Lucia Banci, Ivano Bertini, Claudio Luchinat, and Robert 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 fascinating problem. Spectroscopic studies, particularly through iH 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.