The world’s smallest Rube Goldberg devices are manufactured by Mother Nature. Known as enzymes, these fiendishly complex proteins perform life’s most basic tasks—transforming air and food into tissues and metabolic energy. Like Rube’s ridiculous contraptions, they have parts that grip and bend, swing and rotate. But in place of ball bearings, dominoes, and springs, enzymes use amino acids to accomplish their feats.

How enzymes operate, however, has proven daunting to decipher. For one thing, they’re too tiny to be observed by the most powerful microscopes. For another, they’re constantly in motion, flexing and drifting in the soupy innards of a cell.

Scientists have long relied on static snapshots (x-ray crystallography) and protein sequences to gain insights into enzyme structure and function. But using this information is like reconstructing a car engine from a parts list and a glance below the hood; it’s impossible to understand how it works until you see it running.

Now scientists are doing the next best thing: animating enzymes with models run on supercomputers. Using molecular dynamics modeling, scientists can track the behavior of each of the tens of thousands of atoms that make up enzymes, solvents, and substrates—and watch nature’s microdevices in action.

A tough nut to crack

Among those taking this tack are Zaida Luthey-Schulten, professor of chemistry at the University of Illinois at Urbana-Champaign, and Rommie Amaro. Amaro recently completed her PhD with Luthey-Schulten and is starting a post-doc at the University of California at San Diego. They are using NCSA supercomputers to model the workings of an enzyme that helps manufacture the amino acid histidine. Together with the experimental group of V. Jo Davisson, professor of medicinal chemistry at Purdue University and his student, Rebecca Myers, Amaro and Luthey-Schulten have recently nailed down a key step in the production of amino acids and the building blocks of DNA. Their work made the cover of the July 2005 issue of Biophysical Journal.

“One of the really fabulous things about molecular dynamics simulations is they allow you to see things on an atomistic level. There is no other way to see these types of behaviors right now. And when it’s consistent with the experimental results, you can have really profound insights into these systems,” Amaro says.

The enzyme in their sights has a name as complex as its function: imidazole glycerol phosphate (IGP) synthase. The enzyme “is really the epitome of complexity in enzyme catalysis,” Amaro says. Its job is to make both IGP and AICAR, an ingredient necessary to make DNA and RNA.

Previous research had shown that IGP synthase consists of two parts, hisH and hisF. Each subunit performs half of the enzyme’s duties: hisH transforms the abundant amino acid glutamine into ammonia and glutamate, while hisF uses the ammonia to produce IGP and AICAR.

Though the two subunits might appear to act independently, they always remain docked together when the enzyme is active. The reason behind their close association appears to be ammonia. If released from the enzyme and into the cell, ammonia would instantly react with water and other solvent molecules. Its escape would leave hisF bereft of a substrate. Instead, the scientists realized, ammonia must follow a protected path within the enzyme to travel from hisH to hisF.

Ammonia’s mysterious path

Initial studies suggested the handoff occurred at something resembling a molecular gate. Four strategically placed amino acids appeared to block the entrance from hisH and into the tubelike interior of hisF. Two of the amino acids were positively charged, and two were negatively charged; their strong electrostatic interactions appeared to seal off the mouth of the hisF barrel completely. “Everybody thought that in order for ammonia to make it from one active site to the other, these would have to move aside,” Amaro says.

Previous sequencing studies had indicated that all four of these gate residues were conserved; that is, the same amino acids occupied these positions in both the bacterial and yeast versions of the enzyme. (In biology, conserved structures tend to be critical for an organism to function. They remain unchanged because without them, creatures don’t survive to pass along the defect.)

To determine the role of each gate residue, the scientists replaced them one by one with a generic amino acid and observed what went awry. Checking the experimental and computational results against one another, they reasoned, would narrow down what was actually going on.

One of their substitutions poked a big hole in the gate by substituting a smaller, uncharged amino acid for a bulky, charged one. This nearly derailed the reaction in the laboratory. Normally, the enzyme uses one molecule of glutamine to make one molecule of IGP, an efficient 1:1 substrate/product ratio. The mutation changed the ratio to an abysmal 122:1. “On the computational side, we introduced that same mutation but could really watch the system on an atomistic level. We saw that the water molecules from the solvent rushed into the hole and filled the protected ammonia chamber; it basically flushed the ammonia out,” Amaro says.

A molecular trapdoor

Even more interesting, however, was how the normal, or wild-type, enzyme behaved in the simulation. When the ammonia moved near one of the four gate residues, a lysine, “the lysine actually bent, and ammonia slipped through this newly discovered side opening,” Amaro says.

Once the ammonia had passed inside, the simulation revealed, the lysine swung shut behind it. Once inside, ammonia was forced to remain in the barrel, and water could not chase it out.

Swapping the lysine for a smaller molecule essentially propped this side door ajar. In the laboratory, the reaction’s efficiency slipped from 1:1 to 3:1. The computer simulation showed that while the mutation allowed ammonia to slip into the barrel more easily, it could also diffuse right back out.

“We actually saw the side opening at the beginning and didn’t quite believe it,” Luthey-Schulten says. In earlier simulations, they had pulled a virtual molecule of ammonia through the enzyme using a technique known as steered molecular dynamics (SMD). SMD allows the scientists to recreate events that would take too long—and use up too much computer time—to simulate otherwise. In this case, the researchers used SMD to measure the strength of the chemical bonds ammonia makes with enzyme amino acids at each step of its journey. “By knocking on that gate long enough, we saw a heck of a high barrier, and knew ammonia was not going through that very easily,” Luthey-Schulten says. “A subtle change, and it just went through the side door. We though nah, we must’ve done something wrong.”

In fact, they had discovered the hidden entrance to the kingdom.

SMD also gave the scientists insight into the role of water in the reaction. Water competes with ammonia to bond with amino acids in the barrel’s lining, they found. So having a couple of water molecules in the hisF barrel prevents ammonia from getting stuck. In other words, water helps lubricate the chamber.

A dedicated computing team

The researchers used the NCSA Tungsten cluster to run their 50,000-atom simulations. “NCSA has always been a valuable resource for us; it’s my location of choice. Their system is stable, the programs run very well on it, and the people are just fabulous,” Amaro says. She singles out John Towns, senior associate director of NCSA’s persistent infrastructure directorate, for particular praise. “He’s so responsive to our needs as scientists. If we need dedicated time, he can usually help us out. It’s also really nice to have people you can talk to; those human relationships make a big difference.”

In fact, Amaro and Luthey-Schulten liked working at NCSA so much that they transferred all of their time at another supercomputing center to NCSA.

Thanks to the data-crunching power of supercomputers, Luthey-Schulten says, “this whole field is really coming into its own. We can seamlessly go from bioinformatics to molecular dynamics simulations and energy landscape studies, and learn enough to apply it to other systems very easily.”

She, Amaro, and colleagues have already applied their findings to an even more intriguing mystery about IGP synthase — how molecules bound at opposite ends of the enzyme somehow work together to turn the device on. Their discoveries promise to help biochemists everywhere decipher how organisms build such elaborate enzymatic mousetraps.

This research is supported by the National Science Foundation and the National Institutes of Health.

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Source: NCSA’s Access Magazine. Provided courtesy of the National Center for Supercomputing Applications