Nov. 9, 2001 COLLEGE STATION, November 8 - Enzymes - proteins commonly used to speed up chemical reactions - can render chemical warfare agents and insecticides harmless by breaking them apart. A group of chemists at Texas A&M University is now genetically modifying one of these enzymes, phosphotriesterase, to make it both faster and more selective.
"We know that some natural enzymes have detoxifying properties, so we are trying to make them faster, better and cheaper," says Frank M. Raushel, a professor of chemistry who has been working on enzymes for the last 30 years.
Phosphotriesterase is known for its detoxifying properties of chemical warfare agents such as the nerve agents sarin, soman and VX, as well as agricultural insecticides.
To understand how phosphotriesterase rapidly degrades these compounds, Raushel's team and a team led by led by Hazel M. Holden, professor of biochemistry at the University of Wisconsin-Madison, set about to crystallize the enzyme and determine its structure. Their results, reported in the journal Biochemistry, show that the catalytic part of the enzyme - or "active site" - contains two zinc metal ions.
Raushel and his collaborators have also studied the structure of the enzyme when it binds to a compound. The scientists identified three binding pockets, designated as the "small," "large," and "leaving group" subsites.
"The binding pockets are likely responsible for the orientation of the compounds within the active site of the enzyme," Raushel says. "So we have been attempting to change the sizes and shapes of the binding pockets and look at changes in the enzyme selectivity."
Raushel and his team changed some of the building blocks, or "amino acids," of each pocket one at a time, and replaced them with the amino-acid alanine. Fourteen amino acids were changed (six in the small group, four in the large group and four in the leaving group), creating 14 "mutant" enzymes.
Each of the enzymes was further tested on 10 compounds to see which mutant worked best with which compound and define the corresponding amino acids for any of the three pockets.
For six of the compounds, the two forms in which they appear in nature, which are mirror images of each other but not identical - also called chiral forms - were tested. Indeed, many compounds - notably the military agents - are mixtures of two chiral forms, but the mixture and either form can have important toxicological differences, hence the independent study of each chiral form.
"One of the chiral forms usually fits better than the other one into the active site of the enzyme," Raushel says, "so we can determine which substrate is detoxified faster. Then we can determine whether this effect is enhanced or reversed with mutant enzymes."
The results of these tests, reported in the Jan. 11 issue of the journal Biochemistry, show that amino-acid changes reducing the size of the small group tend to enhance the selection of one chiral form at the expense of the other, and that changes enlarging the large group have a relatively small effect on the selection of the chiral forms.
Raushel is now using new techniques allowing him to make a large number of mutants simultaneously. By further testing each mutant at once on a set of compounds, he can quickly compare the activities of the mutants and select the fastest mutant.
"Whereas it would have taken at least a week to make and characterize a mutant in the past, we can now make 1,000 mutants a day and determine the ones that have enhanced activity," Raushel says.
Each mutant is created by changing a given amino acid into all other possible amino acids. As there are only 20 amino acids in nature, 20 different mutants can be studied simultaneously. But mutants can also be obtained by changing two amino acids at once, increasing the number of possibilities to 400.
Finding the fastest detoxifying enzymes by doing optimal genetic changes is probably within reach. But Raushel admits that he still does not know why some mutants perform better than others for specific compounds.
"We have been able to demonstrate that we can manipulate the enzymes and we can alter their catalytic properties," he says.
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