Principal Investigator Jill Gready Project u53

Division of Biochemistry and Molecular Biology, Machine VP

John Curtin School of Medical Research

Co-Investigators William King and John Andrews

Division of Biochemistry and Molecular Research, John Curtin School of Medical Research and Research School of Biological Sciences.

Definition of the Chemical Mechanism of the Photosynthetic Enzyme Rubisco

The project is part of a new collaborative project between JCSMR and Research School of Biological Sciences (RSBS). The project is a collaboration with Professor John Andrews in the Research School of Biological Sciences who has wide-ranging interests in the enzyme Rubisco. These include undertaking biophysical studies and enzyme kinetics on native and mutant forms to understand the chemical mechanism, and identification and structural characterisation of reaction byproducts and intermediates. It is expected that the project will lead to predictions which could be tested experimentally.

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.

What are the basic questions addressed?

There are a very large number of structure/function questions for Rubisco which might be studied. The aim is to start with two aspects of the reaction mechanism which appear from the literature to be critical and for which the Andrews' group has experimental interest and expertise for followup. The highly reactive chemistry which takes place sequestered from bulk water suggests that comparative studies of the structures, properties and possible reaction pathways for the proposed reactive species and how these are affected by the active-site might aid understanding of how the proposed active-site compromise is organized. (Similar studies are being undertaken for the much simpler mechanism of lactate dehydrogenase.) There appear to be conflicts in the requirements of the active site to stabilise the different species and this study might start to clarify them. It is proposed to start with the enolization which produces the 2,3-enediol form which is carboxylated, normally, or oxygenated, wastefully.

The second specific question relates to the oxygenase reaction and in particular how molecular oxygen, which is triplet in the ground-state, reacts with bound ground-state singlet substrate such as RuPB to produce singlet products.

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

Several X-ray structures of different forms of the enzyme and complexes are available for construction of active-site models. Some preliminary MD simulations have been performed using a number of these initial X-ray structures in order to define the possible conformations of the reactive species in the active site, and provide starting coordinates for studies on the reactive complexes.

What computational techniques are used and why is a supercomputer required?

The computational part of the project consists of semiempirical and ab initio quantum chemical studies of substrates, intermediates, and small enzyme ligand complexes, and molecular dynamics (MD) and QM/MM (semi-empirical quantum mechanics/molecular mechanics) calculations on the enzyme-bound species. The MD and quantum chemical calculations are performed using Amber 4.1 and GAUSSIAN 94 vectorised for the VP2200. Programs being developed in the Group by Dr Peter Cummins under VP project u51 will be used for the QM/MM calculations