Stop-action crystallography tracks enzymes.

Thanks to those ever-adapting soil bacteria, nature has come up with its own ways to get rid of pollutants (SN: 8/15/92, p. 107; 3/14/92, p. 175). Some microbes, for example, have evolved enzymes that break down chlorinated compounds into less toxic components.

Now a Netherlands research team seeking to understand how these enzymes work and to improve upon nature's cleanup efforts has caught one in the detoxification act.

The investigators manipulated the temperature and acidity of solutions containing crystals of haloalkane dehalogenase from a nitrogen-fixing bacterium, Xanthobacter autotrophicus. This slowed the speed of the reaction typically much faster than a second -between the enzyme and its substrate. This enabled them to use X-ray diffraction to determine the positions of the atoms at different stages of the reaction. Koen H.G. Verschueren and his colleagues at the University of Groningen describe their results in the June 24 NATURE.

The data confirm that the enzyme breaks up the pollutant in two steps, not one, as some researchers have suggested, says Bauke W. Dijkstra, a crystallographer with the group.

The team first placed enzyme crystals in an acidic solution (pH 5) with l,2-dichloroethane, cooling it to 4degreeC. Under those conditions, the chlorinated molecule binds to the enzyme but no reaction occurs. Warming the solution to room temperature, allowed the enzyme to break the bond between one chlorine and a carbon atom of the molecule. Finally, making the solution less acidic (pH 6.2) pushed the reaction further.

The three stop-action atomic structures they obtained suggest that the chlorinated molecule winds its way through a narrow channel in the enzyme to the active site, an isolated internal pocket where the reaction occurs. Once there, it encounters a water molecule that the amino-acid side chains use to detoxify the incoming molecule.

During the first step, a chloride ion breaks away, allowing the rest of the molecule to link with an amino acid as an ester molecule, Dijkstra explains. The negatively charged ion helps make another nearby amino acid more able to attract a positive hydrogen from one water molecule. In the second step, that disrupted water molecule turns the ester into an alcohol, which is then released from the active site. The chloride leaves last.

"The problem is that this enzyme is not very active," says Dijkstra. "It's a relatively young enzyme; in an evolutionary sense, there hasn't been enough time for the bacteria to adapt."

Although eventually the enzymes would become more efficient, he and his colleagues hope to make them work faster and process more kinds of contaminants by manipulating the bacteria in the laboratory, "If we can understand the mechanism, then we may be able to improve the activity," he says.

Dijkstra expects others will try this crystallographic approach to study the activity of other enzymes.