Definition of the Chemical Mechanism of the Photosynthetic Enzyme Rubisco

Principal Investigator

Jill Gready

Division of Biochemistry and Molecular Biology

John Curtin School of Medical Research

Rubisco (D-ribulose 1,5-bisphosphate carboxylase-oxygenase), the enzyme catalysing the fixation of CO2 in photosynthesis, is arguably the most important enzyme, and is also the most abundant protein, on earth. The great puzzle is why Rubisco is still such an incompetent enzyme despite predictably extreme evolutionary pressures. Many aspects of the enzyme's complex structure and function suggest it is a compromise solution to effecting quite difficult chemistry as the proposed reaction sequence involves as many as four chemically-unstable enzyme-bound intermediates. The slow catalytic rate, poor selectivity for binding of its substrate CO2 compared with O2 and the existence of significant side-reactions indicate both activation and control of the component reactions are very poor. While the role of enzymes in facilitating reactions by substrate activation and reaction rate enhancement is well acknowledged, their role in selecting reactions from possible reactions in the metabolic "soup" and channelling them so as to avoid unwanted products has received little attention. This latter aspect appears especially important for Rubisco and the proposed studies are aimed at understanding how the enzyme active site is designed to maximise both these aspects, which may be incompatible, and thus to understand in what ways the active site design may be a compromise. While there are a very large number of structure/function questions which might be studied, we have started with the initial enolization step which produces the 2,3-enediol form which is carboxylated, normally, or oxygenated, wastefully.

Co-Investigators

Bill King

John Andrews

Division of Biochemistry and Molecular Biology

John Curtin School of Medical Research

& Research School of Biological Sciences

Projects

u53 - VPP, PC


What are the results to date and the future of the work?

As a first step to constructing starting co-ordinates for fragment "supermolecule" calculations at the ab initio quantum mechanical (QM) level and for QM/MM calculations, we needed to generate structures for relevant enzyme complexes. Those of immediate interest, i.e. the active RuBP and enol-RuBP complexes, are, of course, not directly available from experimental x-ray structures. There are few good x-ray structures available for close analogues to substrate, the best being a 1.6Å structure for spinach Rubisco with bound CO2, Mg and CABP (2-carboxy-

- Appendix A



arabinitol-1,5-bisphosphate). After several attempts to substitute ligand coordinates for an active RuBP complex and relax it with MD we succeeded in obtaining a satisfactory set of relaxed co-ordinates for the substrate complex. These have been used so far in "supermolecule" fragment calculations.

The first aim of understanding the enolization of RuBP requires investigating how the enolization is promoted in the enzyme active site. By calculating the zero point energies along a proposed reaction pathway, we can investigate the role that different groups have on the reaction, by observing the effect that removing them has on the activation energy. Using coordinates produced by the MD calculations, we have undertaken ab initio QM calculations on a 29 atom fragment of the active site at several basis set levels. Six species (reactant, product, transition and intermediate states) for a proton hopping mechanism are being investigated. Preliminary results suggest that the novel reaction model we are examining is possible.

What computational techniques are used?

Molecular dynamics and quantum mechanical calculations have been undertaken with AMBER 4.1 and GAUSSIAN 94, which are vectorized for the VPP300.