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Molecules Caught in the Act-- Scientists at The Scripps Research Institute Solve Elusive Enzyme Mechanism

La Jolla, CA, October 12, 2001-- A collaboration between Professors Ian A. Wilson, Ph.D., Chi- Huey Wong, Ph.D., and their colleagues in the Departments of Chemistry, Molecular Biology, and The Skaggs Institute for Chemical Biology at The Scripps Research Institute (TSRI) has yielded one of the best views ever of an enzyme caught in the act of catalyzing a reaction on its substrate. This research should prove invaluable as a tool for drug synthesis.

In the current issue of the journal Science, the team has published a report showing the intermediate structures of the enzyme D-2-deoxyribose-5-phosphate aldolase (DERA) and its substrates. DERA is one of the sugar-metabolizing class-1 aldolase enzymes.

"This is the best high-resolution structure I have seen in structural enzymology," says Wong, "It allows us to further design experiments to define the detailed mechanism atom by atom."

In fact, the structures were solved at 1.05 and 1.10 angstrom resolutions-- fine enough to discern objects that are one ten-millionth of a millimeter long, including individual atoms in the DERA molecule.

What's more, because the report details the intermediate structures of DERA and substrates, the description is more than just a physical mechanism-- it is a direct image of the enzyme in action. "This is the most complete mechanism that has been published of an aldolase," says Wilson.

Biochemical catalysis involves using proteins to greatly enhance the speed and efficiency of a specific chemical reaction-- like breaking or forming a bond. Sometimes enzymes catalyze these reactions by changing their substrates a little at a time, taking them through one or more intermediate states.

Normally, studies of these intermediate states are hindered by a lack of detailed structural information. Techniques like x-ray crystallography, in which crystals grown in concentrated solutions of the enzyme are bombarded with x-rays, diffract in a pattern corresponding to the final structure of the enzymes in the crystal.

However, Andreas Heine of the Wilson laboratory was able to soak the substrate into the crystals, allow the reaction to proceed to its intermediate state, and then quickly flash freeze the crystals in liquid nitrogen to trap them in that state-- like a snapshot of two tango dancers in mid- twirl.

Mutating particular amino acids in the active site of DERA allowed Wong to make two different forms of the enzyme with distinct intermediates in the reaction. This led to the identification of a critical water molecule close to the active site that the enzyme uses as a mediator of the interaction.

These structures should enable Wong to broaden DERA's use in making important intermediates for pharmaceutical synthesis-- the basis for the industrial preparation of commercial drugs.

"They will be a guide to help us engineer this enzyme to alter or improve its specificity," says Wong.

The research article, "Observation of Covalent Intermediates in an Enzyme Mechanism at Atomic Resolution" is authored by Andreas Heine, Grace DeSantis, John G. Luz, Michael Mitchell, Chi-Huey Wong, and Ian A. Wilson and appears in the October 12, 2001 issue of the journal Science.

The research was funded in part by the National Institutes of Health and The Skaggs Institute for Research.


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