Enzyme channeling is the directed transport of substrate intermediates between active sites on an enzyme. Channeling increases the efficiency of catalysis tenfold or more. Below we describe two enzymes from nitrogen metabolism that are well-characterized examples of enzyme channeling.
Tryptophan synthase catalyzes the last two steps in the tryptophan synthesis pathway. The first step generates indole (the double-ring portion of tryptophan's side chain) from indole-3-glycerol phosphate (a molecule much smaller than its name). In the second step, the substrate amino acid serine receives the indole, which is added on to its side chain. This second step essentially transforms serine into tryptophan.
The two enzymatic activities of tryptophan synthase are found on two separate subunits in bacteria, and on a homologous single polypeptide chain in yeast and some other eukaryotes. The bacterial enzyme functions as a tetramer, (ab)2. For simplicity, we'll use a single ab dimer from the bacterium Salmonella as a model. Each subunit bears one active site (yellow); they are about 25 Å apart.
Note that to see behind any surface rendering, you can use the Chime menu: Select–Display List–Toggle Visibility (or Toggle Transparency).
Indole-3-glycerol phosphate (IGP), the substrate for the first reaction, binds in the a subunit active site. Shown here is a structurally similar inhibitor of the enzyme, indole propanol phosphate (IPP).
Binding of a substrate to an enzyme usually triggers catalytic activity, making it difficult to study enzyme structure with a bound substrate. One way to get around this difficulty is to use an inhibitor of the enzyme. An inhibitor that structurally resembles the substrate can bind correctly in the active site. The small difference in structure, however, renders the enzyme unable to act—so the enzyme can be studied with this bound "substrate analog." (It is then important to perform additional studies to confirm that the structure of the enzyme is not altered by the inhibitor, nor is it altered in the same way as when the true substrate binds.)
The inhibitor indole propanol phosphate (IPP) is used to pinpoint and study the active site of tryptophan synthase, which normally binds the substrate indole-3-glycerol phosphate (IGP). Compare the two structures.
The first reaction, the conversion of indole-3-glycerol phosphate to indole, is catalyzed by the a subunit of trytophan synthase. Without indole-3-glycerol phosphate bound in the a active site, a loop of the a subunit (Loop 6) flops around, leaving the substrate binding site open. The structure of Loop 6 cannot be determined while the loop is mobile. When indole-3-glycerol phosphate binds in the a active site, a dramatic conformational change occurs: Loop 6 (orange) is stabilized and covers the active site. This traps the substrate and prevents it from diffusing away. When Loop 6 is unstable, the enzyme is less active than when the loop is stabilized. Among other things, this indicates that without this "lid," the substrate can be easily lost to the surrounding solution.
Once indole-3-glycerol phosphate is bound in the a subunit, it is converted to indole by cleavage of the bond (flashing) between the glycerol phosphate moiety (white) and the indole moiety.
The second reaction takes place in the active site of the b subunit. Here, a pyroxidal 5'-phosphate (PLP) prosthetic group temporarily forms a covalent bond with the substrate serine. The pyroxidal 5'-phosphate holds the serine in place for the addition of the indole rings that convert serine to tyrosine. The rings are added in place of serine's side chain, which is an OH group (shown as an oxygen atom since hydrogen atoms do not appear in X-ray crystallography). Following the addition reaction, the bond linking tyrosine to the pyridoxal 5’-phosphate group is cleaved, and tyrosine is released from the enzyme.
It is 25 Å from the a active site to the b active site. How does the indole intermediate make this long trip? Kinetic studies show that the indole produced in the a subunit active site reaches the b subunit active site in record time (less than 1/1,000 of a second)! This and other evidence strongly suggests that indole does not randomly diffuse to the b site, rather it travels by an internal tunnel.
The protein has been slabbed approximately half-way through and some residues have been removed in this view to make the tunnel visible. Experiment with the slab to uncover and cover the inside of the protein (control–click–drag).
It is important to remember some of the fundamentals of molecular structure when looking at a tunnel in a protein. Proteins are not static molecules, despite their solid, motionless appearance. They vibrate, and flex, and some loops flop about. As a result, any snapshot of a protein (such as an X-ray crystal structure) only tells part of a story. Under some circumstances, tunnels are blocked. For example, a few flexible side chains are known to move in and out of the tryptophan synthase tunnel. So the structures of the tunnels shown in this tutorial may differ slightly from their structures in the active enzymes.
The interiors of soluble proteins are generally packed solidly with nonpolar residues. There are small cavities here and there, but the presence of a tunnel of any appreciable length is highly unlikely to occur by chance. The fact that the tunnel has survived natural selection (recall that tryptophan synthase is conserved in bacteria and eukaryotes) strongly implies that the tunnel is necessary for the function of the enzyme. In addition, the tunnel is predominantly lined with hydrophobic groups (green), which are compatible with the aromatic indole group that must pass through.
Tryptophan synthase is not the only enzyme that employs a channeling strategy. We now turn our attention to a particularly complex and exquisite enzyme, carbamoyl phosphate synthetase.
Carbamoyl phosphate is a nitrogen-containing compound that is essential for two critical metabolic pathways: the urea cycle and pyrimidine nucleotide synthesis. Carbamoyl phosphate is built using the amino acid glutamine, bicarbonate (HCO3–), and two molecules of ATP. The reactions also produce glutamate, ADP, and phosphate.
In E. coli, carbamoyl phosphate synthetase is a heterodimer of a large and small subunit.
The enzyme has three active sites that are a considerable distance apart.
|1)||The glutamine amidotransferase site removes ammonia (NH3) from glutamine.This reaction requires the formation of a thioester bond between the glutamine substrate and a cysteine residue in the active site. In this view, the backbone of the glutamine substrate is at the opening of the site, and the side chain extends deep into the binding pocket, where it has already formed a thioester bond with cysteine. The glutamine NH3 group has already been extracted.|
|2)||The carboxyphosphate site uses HCO3– and ATP to make carboxyphosphate, which then reacts with the NH3 extracted from glutamine to make a reactive compound called carbamate. ADP, which is a product of this reaction, is shown in the active site.|
|3)||The carbamoyl phosphate site uses carbamate and a second molecule of ATP to make carbamoyl phosphate.|
All three active sites of E. coli carbamoyl phosphate synthetase are visible in the next display. Show the distance between any two sites by clicking on an atom in or near a site, and then clicking on an atom in or near a second site. You can read the distance in angstroms in the "picked atom" output text in the title bar. You can also display the distance of these pairs of atom clicks as a label directly on the molecule by toggling the distance monitor in the Chime menu (Select–Mouse click Action–Toggle Distance Monitor). Note that the substrates must travel from site 1 to site 2 and then on to site 3. Calculate the total distance the substrates must travel to produce carbamoyl phosphate.
As you may have guessed by now, substrates traveling these distances by diffusing from site to site could easily be lost, or, given the reactivity of the intermediates in these reactions, they could react with other molecules to form compounds that are useless for building carbamoyl phosphate. For example, NH3, the product of the first reaction, readily extracts a proton from water to become NH4+. Since the ammonium ion cannot react in site 2, the substrate would be wasted. Similarly, the carbamate produced in reaction 2 is a reactive molecule that could be lost to the surroundings. A channel, however, could shelter these intermediates. Evidence strongly suggests that carbamoyl phosphate synthetase, like tryptophan synthetase, channels substrates from site to site, using two internal tunnels:
The residues that have been removed (faded out) to reveal the tunnels are those that overlay them most directly. The yellow surface represents the far side of the tunnel from the viewer, defining an approximate path between the sites. Take the time to explore each tunnel by zooming (shift-drag), translating (Mac, option-drag; PC, control–drag), and rotating the structure. Note that the second tunnel is wider than the first. Why do you think this is so?
The presence of channels in multifunctional enzymes such as tryptophan synthase and carbamoyl phosphate synthetase maximizes metabolic efficiency. As more enzyme structures are solved, it seems likely that we will find more examples of tunnels and other means of controlling the passage of intermediates.
To review the animation of intermediates moving through the tunnel of carbamoyl phosphate synthetase, press the button below:
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