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Using computers to unlock nature's secrets

Model of methylmalonyl-CoA mutase, a coenzyme
B12 dependent enzyme.

Sophisticated computer simulations performed by the University of Sydney node of the Free Radical Centre are adding powerful insight into how a particular class of enzymes catalyse reactions.

Life relies on the ability of enzymes to catalyse complex reactions efficiently and with high fidelity. In order to catalyse a reaction, an enzyme undergoes a myriad of structural changes, which ultimately results in the conversion of the reactant (starting compound) to the desired product. Understanding the mechanisms of these processes may help us to design new, more robust catalysts, or even to prevent unwanted side reactions that sometimes occur in the natural cycles of these processes. For example, because of the industrial importance of glycerol, a significant amount of effort has been invested in enhancing the efficiency of the enzyme-catalysed transformation of this compound in order to reduce costs.

Our knowledge of these processes has been traditionally obtained within experimental laboratories where intricate biochemical, kinetic, and spectroscopic experiments are performed.  While these are undoubtedly crucial elements to achieve success in understanding how enzymes function, they are often time-consuming, costly and environmentally unfriendly.  However, the present age of high performance supercomputers and sophisticated algorithms is providing an attractive and insightful adjunct to these conventional methods.

Together with his post-doctoral fellow Dr Greg Sandala, Professor Leo Radom at the School of Chemistry at the University of Sydney and the ARC Centre of Excellence for Free Radical Chemistry and Biotechnology, and long-time colleague Dr David M. Smith from the Ruðer Boškoviæ Institute (Zagreb, Croatia), a partner institute, are using computer simulations to explore the relationship between structure and function in enzymes at an atomic level. The simulations do this by exploring the shape (or conformation) of the enzyme in the presence of the reactant, and determine various pathways that the reactant can take to reach the final product. The pathway that requires the least amount of energy is usually considered to be the most probable, and by examining the details of these pathways, scientists can then identify the origin of an enzyme's catalytic capability.

A class of enzymes suitable for computer simulation study are those reliant on coenzyme B12, the so-called coenzyme B12-dependent enzymes. Coenzyme B12 is one of two biologically active forms of vitamin B12. In humans, coenzyme B12 is used by methylmalonyl-CoA mutase (MCM) in the catabolism of odd-chain fatty acids, branched-chain amino acids, and cholesterol. The product of this reaction, i.e., succinyl-CoA, can then be used as a metabolite for other important processes, such as the citric acid (or Krebs) cycle used in energy generation. Deficiencies in the operation of MCM lead to the autosomal recessive disorder methylmalonic aciduria.

A remarkable feature of these enzymes is their purpose-built capacity to generate free radical intermediates. Free radicals are normally considered harmful to biological systems on account of their rapid and indiscriminate reactivity. In this case, however, coenzyme B12-dependent enzymes actually generate them on purpose! For this reason, these enzymes represent an excellent model from which to gather information on how enzymes control and manipulate high-energy intermediates in order to catalyse difficult chemical transformations. Moreover, these reactions comprise fundamental steps in basic processes such as energy generation and DNA replication and repair. Computer simulations thus offer unprecedented insight into these processes, and provide information that would otherwise be difficult to obtain in the laboratory.

Two questions that have been rigorously debated concerning the reactions catalysed by coenzyme B12-dependent enzymes is how these enzymes convert their reactants to products, and why this process sometimes fails irreversibly, a phenomenon termed suicide inactivation. So far the results have shown that unique amino acids within the active site of the enzymes assist the conversion of reactant to product via very specific hydrogen bonds. With certain substrates, however, irreversible inactivation appears to result from the generation of exceptionally low energy free radical intermediates that terminate the catalytic cycle prematurely because they are too stable to react any further.

Another long-standing question concerning these enzymes is how they activate the enzyme bound coenzyme B12 by up to one trillion times compared with the free coenzyme in solution. The Sydney group is seeking to address this fascinating display of enzyme prowess with large scale molecular dynamics that run in parallel over several hundred computer processors. Their preliminary data suggest that this exceptional activity results from a sequence of tightly controlled and coordinated interactions, which combine to produce the overall activation of coenzyme B12.

Such atomic level insights are now possible because of the advancements in computer hardware and software. In the future, computer modelling and simulations like these will undoubtedly play an increasingly prominent role in unlocking the secrets of enzyme catalysis – a task once confined to the laboratory.