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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.
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