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Theory meets experiment: a combined quantum chemical-experimental study of the reaction mechanism of pI258 arsenate reductase

Wednesday, 2 May, 2007 - 11:00
Campus: Brussels Humanities, Sciences & Engineering campus
Faculty: Science and Bio-engineering Sciences
auditorium Q.b
Goedele Roos
phd defence

Although studied for decades, enzymatic catalysis remains one of the most intriguing biochemical phenomena. A remarkable example of a catalytic mechanism has been documented for pI258 arsenate reductase (ArsC). This enzyme combines a unique disulfide cascade mechanism involving Cys10, Cys82 and Cys89 with the functional unfolding of a flexible redox helix. This study was started to unravel all the subtle, important details used by pI258 ArsC to reduce arsenate to arsenite. Disentangle means digging into the heart of the enzymatic reaction mechanism. The limitations of today’s known biochemical approaches made us to use quantum chemical tools. Central in quantum chemistry is the Schrodinger’s time independent equation HΨ=EΨ with Ψ the wave function and E the energy. For many electron systems, several approximation methods are available to obtain Ψ and E out of this equation, and from Ψ, a variety of molecular properties (for example the electron density function, atomic charges). Throughout this work, Density Functional Theory (DFT) is used as a tool to calculate these properties. Among other things, these molecular properties have taught us that subtle changes of the structural environment of ArsC determine the probability of a cysteine residue to function as a nucleophile.

More in detail, in this thesis, we focussed on the onset of the nucleophilic attack of Cys10 on arsenate, leading to a covalent Cys10-arseno intermediate, and of Cys82 on this adduct. Additionally, the nucleophilic attack by Cys89 on Cys82 leaving ArsC in its oxidized form at the end of a single catalytic cycle, is studied. The central questions are: ‘Is arsenate bound as mono- or as di-anion in the Michaelis complex?, ‘Is the covalent Cys10-arseno intermediate mono- or di-anionic? and ‘How does ArsC activate the leaving groups (water and arsenite) and the nucleophiles (Cys10, Cys82 and Cys89) in the reactant state?’

Further, thioredoxin regenerates arsenate reductase in its reduced form for a subsequent catalytic cycle. An answer is given on the question ‘What makes thioredoxin a reducing agent?’ Here, the focus goes to the intriguing role of the highly conserved proline in the active site of this ubiquitous redox enzyme.

The strength of the presented work is in the multidisciplinary approach. All essential intermediates in the reaction mechanism of ArsC are available, providing a unique data-set of high-resolution X-ray structures. These structures offer the opportunity to perform theoretical studies via model systems, giving insight in problems which are experimentally difficult to access. Moreover, when applicable, our theoretically obtained results are discussed in the light of experimental data. As such, the interplay between theory and experiments has permitted us to gain full insight into the enzymatic reaction mechanism of pI258 ArsC.