Study of Intramolecular Electron Transfer and ...

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Department of Biochemistry, University of. Leicester ... Biochemistry, Ohio State University, Ohio, USA ... Vilnius University, D e p m e n t of Analytical Chemistry.
A46

Biochemical Society Transactions (1999) 27

57

Substrate inhibition in wild-type and mutant trimethylamine dehydrogenases

Peter Roberts, Jaswir Basran, Martin Mewies, Russ Hille and Nigel S. Scrutton Department of Biochemistry, University of Leicester, UK and Department of Medical Biochemistry, Ohio State University, Ohio, USA The reduction kinetics of wild-type and mutant forms of trimethylamine dehydrogenase using trimethylamine and a variety of alternative tertiary amine substrates have been studied by rapid-mixing stopped-flow spectroscopy and steady-state kinetic analysis. The data reveal that the extent of substrate inhibition varies with different substrate/wild-type combinations and with selected mutant forms. Multiple wavelength rapid-mixing stopped-flow analyses of wild-type and mutant forms of trimethylamine dehydrogenase were used to address the mechanism of substrate inhibition. Results of these studies will be presented.

59 Structure and mechanism of an opiate-

transforming redox enzyme: morphinone reductase Daniel H. Craig, Neil C. Bruce, Peter C. E. Moody and Nigel S. Scrutton Department of Biochemistry, University of Leicester, University Road, Leicester UK and Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, UK The structure of morphinone reductase has been solved at 2.5 A resolution revealing a dimer comprising two subunits folded as an eight-fold pla barrel, each responsible for binding flavin mononucleotide. The mechanism of morphinone reductase was also studied by rapid-mixing stopped-flow spectroscopy. Reduction of the flavin by NADH involves formation of a charge-transfer complex, followed by flavin reduction. Oxidation of the enzyme by codeinone proceeds in three kinetically resolvable steps; charge-transfer formation, flavin oxidation and product release. A detailed kinetic and thermodynamic analysis of the enzyme will be presented.

60

Study of IntramolecularElectron Transfer and Catalytic Action of Quinohemoprotein-Alcohol Dehydrogenase from Gluconobacter sp. 33

Aninas RamanaviEiusl.2,Julija Razumiene*’, Valdas LaurinaviEius2,Liucija

MarcinkeviEiene’, Irina Bachmatova’, Rolandas MeSkys’, Rolandas Rudomanskis’. 1. Vilnius University, D e p m e n t of Analytical Chemistry. Naugarduko 22, Vilnius, Lithuania 2. Institute of Biochemistry, Laboratoryof Bioanalysis, 2600 Vilnius, Lithuania,

58 Electron transfer in o-hydroxylation: analysis of rubredoxin reductase and rubredoxin

Ho Joon Lee, Jaswir Basran, Lu-Yun Lian and Nigel S. Scrutton Department of Biochemistry, University of Leicester, University Road, Leicester UK Rubredoxin reductase and the 2Fe-rubredoxin from Pseudomonas oleovorans have been cloned, expressed to high levels and purified from recombinant strains of Escherichia coli. The electron transfer properties of this redox system have been investigated using a combination of rapid-mixing stopped-flow spectroscopy, redox titration and structural nmr methods. A method for converting the paramagnetic 2Fe-rubredoxin into the cadmium-substituted, diamagnetic form has been developed, thus enabling structural work by nmr. A study of the redox, kinetic and spectroscopic properties of the two proteins will be presented.