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Copying nature's chemistry
T. Daniel P. Stack is in the business of mimicking nature.
The assistant professor of chemistry builds miniature working models of enzymes, the proteins that life uses to speed chemical reactions.
"It's a matter of seeing what nature has done and then asking, 'Can we do what she does? And can we do it in a simpler way?' " Stack says.
His recent work answered those questions with a resounding yes. Earlier this year, Stack and his colleagues reported in the journal Science that they have designed, synthesized and characterized a molecule that looks like an enzyme and acts like an enzyme but is a fraction of its size. This catalyst is one of only a handful of working models of enzymes created to date.
In this case, smaller is better because small molecules, like Stack's creation, are inherently easier to study than large ones, like enzymes. The model enzyme even may have industrial potential because it is easy to work with and it catalyzes an important organic reaction without making polluting byproducts.
Enzymes play a crucial role in life's chemistry. They speed chemical reactions, coming away unchanged and ready to start again. Enzymes catalyze such reactions as the breakdown of the air's nitrogen, an important plant nutrient, into a form that plants can use. Other enzymes catalyze the transformation of starch to sugar, and still others regulate protein synthesis in the body.
Chemists dream of catalyzing reactions the way that enzymes do: working at room temperature and low pressure, using simple materials like nitrogen to make only useful products and benign byproducts like ammonia and water. The only ways that researchers can currently duplicate many of these reactions are by applying extremely high temperatures and pressures, or by using exotic (and often toxic) metals.
One of the obstacles to understanding enzymes is their large size. Each enzyme comprises thousands of atoms, with various sections of an enzyme performing different functions. At a specific location within the enzyme, called the "active site," reacting molecules come together, break apart and recombine as they interact with the enzyme, and then leave as different molecules. Other sections of the enzyme can block the active site, allowing only certain molecules access or permitting approaches to the active site from only one direction.
Stack chose a well studied and structurally characterized enzyme, galactose oxidase, to mimic. That way, he reasoned, he had a good chance of integrating the important structural features into his model. Galactose oxidase catalyzes the reaction of alcohols (organic molecules containing an oxygen bound to a hydrogen) with oxygen gas.
"Chemistry is chemistry. If a big molecule can catalyze a reaction, a small molecule might be able to do it too," says Yadong Wang, the graduate student working on the project in Stack's lab.
To prove that one of life's chemical reactions could be duplicated and even simplified in the lab, Stack and Wang eliminated thousands of extraneous atoms from the enzyme, but retained a structure similar to the enzyme's active site. Their catalyst consists of a copper atom surrounded by two nitrogens and two oxygens, which are connected by a carbon-and-hydrogen scaffold.
Stack and Wang avoided one problem that had thwarted many chemists' previous attempts to build small enzyme models; they prevented the model molecules from interacting with each other instead of with oxygen and alcohol molecules as they are supposed to by making the scaffold sufficiently bulky.
Once synthesized, the model had to pass several tests before the researchers were convinced that it was a good mimic of the enzyme:
Stack and Wang mixed together their enzyme mimic with an alcohol and added oxygen gas to determine whether their molecule catalyzed the oxidation of alcohols as galactose oxidase does. They found that their model functioned as a catalyst each model molecule aided more than 1,000 alcohol molecules in reacting with oxygen like the natural enzyme.
To measure its structure and spectra, and to see how the alcohol molecule interacted with the mimic's active atoms, Stack and Wang sought the help of spectroscopists at the Stanford Synchrotron Radiation Laboratory: chemistry graduate student Jennifer DuBois, chemistry Professor Keith Hodgson and senior research associate Britt Hedman.
Using X-ray absorption spectroscopy, DuBois, Hodgson and Hedman determined the structure of the enzyme model in different chemical states, showing that the small molecule took on three-dimensional forms similar to the enzyme's active site.
This analysis also showed that the enzyme mimic led the alcohol molecule through the same series of intermediates that scientists believe galactose oxidase does.
Stack's catalyst passed all three tests, making it a rare example of small working models of enzymes.
Not only are such small molecules easier to analyze and easier to purify than large ones, but they are also simpler to alter, for instance, to remove a certain atom or group of atoms to test its importance in the catalyst's activity. "Now you're really in a position to test any hypothesis about this chemistry that you want," DuBois says.
Of course, some of the conclusions drawn from studying a model may not hold for the enzyme itself. Similarly, the groups of atoms that Wang and Stack found were important to include in their model may not necessarily play the same role in the enzyme.
"It's indirect proof," Wang says. "The more indirect proof you have that points in the same direction, the more likely it is that your hypothesis is right."
Manipulating the enzyme mimic could even transform it into an industrially useful catalyst. Galactose oxidase and Stack's model catalyze alcohol oxidation, an important reaction in syntheses of many pharmaceuticals and other organic molecules. At present, the model catalyst is too slow and stops working too soon to make it industrially useful. But tweaking the catalyst and reaction conditions (such as temperature and whether the reaction is run in water or alcohol or some other solvent) could make the enzyme mimic into a commercially viable catalyst.
Stack says that the advantage nature's methods provide over the ones chemists now use is that the reactants are cheap they often come from the air and the byproducts are benign.
"If we lived in a world that said, 'You can generate only minimal toxic waste,' then these catalysts would become very important," Stack says. Stack and his colleagues received funding from the National Institutes of Health, the National Science Foundation and the Department of Energy.
By Lila Guterman