Certain amino acid side chains of an enzyme are important in determining its specificity and catalytic power. In the native conformation of an enzyme, these side chains are brought into proximity, forming the active site. Active sites thus consist of two functionally important regions: one that recognizes and binds the substrate (or substrates) and another that catalyzes the reaction after the substrate has been bound. In some enzymes, the catalytic region is part of the substrate-binding region; in others, the two regions are structurally as well as functionally distinct.
To illustrate how the active site binds a specific substrate and then promotes a chemical change in the bound substrate, we examine the action of cyclic AMP-dependent protein kinase, now generally referred to as protein kinase A (PKA). This enzyme and other protein kinases, which add a phosphate group to serine, threonine, or tyrosine residues in proteins, are critical for regulating the activity of many cellular proteins, often in response to external signals. Because the eukaryotic protein kinases belong to a common superfam-ily, the structure of the active site and mechanism of phos-phorylation are very similar in all of them. Thus protein kinase A can serve as a general model for this important class of enzymes.
The active site of protein kinase A is located in the 240-residue "kinase core" of the catalytic subunit. The kinase core, which is largely conserved in all protein kinases, is responsible for the binding of substrates (ATP and a target pep-tide sequence) and the subsequent transfer of a phosphate group from ATP to a serine, threonine, or tyrosine residue in the target sequence. The kinase core consists of a large domain and small one, with an intervening deep cleft; the active site comprises residues located in both domains.
Substrate Binding by Protein Kinases The structure of the ATP-binding site in the catalytic kinase core complements the structure of the nucleotide substrate. The adenine ring of ATP sits snugly at the base of the cleft between the large and the small domains. A highly conserved sequence, Gly-X-Gly-X-X-Gly-X-Val (X can be any amino acid), dubbed the "glycine lid," closes over the adenine ring and holds it in position (Figure 3-17a). Other conserved residues in the binding pocket stabilize the highly charged phosphate groups.
Although ATP is a common substrate for all protein ki-nases, the sequence of the target peptide varies among different kinases. The peptide sequence recognized by protein kinase A is Arg-Arg-X-Ser-Y, where X is any amino acid and Y is a hydrophobic amino acid. The part of the polypeptide chain containing the target serine or threonine residue is bound to a shallow groove in the large domain of the kinase core. The peptide specificity of protein kinase A is conferred by several glutamic acid residues in the large domain, which form salt bridges with the two arginine residues in the target peptide. Different residues determine the specificity of other protein kinases.
The catalytic core of protein kinase A exists in an "open" and "closed" conformation (Figure 3-17b). In the open conformation, the large and small domains of the core region are separated enough that substrate molecules can enter and bind. When the active site is occupied by substrate, the domains move together into the closed position. This change in tertiary structure, an example of induced fit, brings the target peptide sequence sufficiently close to accept a phosphate
▲ FIGURE 3-17 Protein kinase A and conformational change induced by substrate binding. (a) Model of the catalytic subunit of protein kinase A with bound substrates; the conserved kinase core is indicated as a molecular surface. An overhanging glycine-rich sequence (blue) traps ATP (green) in a deep cleft between the large and small domains of the core. Residues in the large domain bind the target peptide (red). The structure of the kinase core is largely conserved in other eukaryotic protein kinases. (b) Schematic diagrams of open and closed conformations of the kinase core. In the absence of substrate, the kinase core is in the open conformation. Substrate binding causes a rotation of the large and small domains that brings the ATP- and peptide-binding sites closer together and causes the glycine lid to move over the adenine residue of ATP thereby trapping the nucleotide in the binding cleft. The model in part (a) is in the closed conformation.
group from the bound ATP. After the phosphorylation reaction has been completed, the presence of the products causes the domains to rotate to the open position, from which the products are released.
The rotation from the open to the closed position also causes movement of the glycine lid over the ATP-binding cleft. The glycine lid controls the entry of ATP and release of ADP at the active site. In the open position, ATP can enter and bind the active site cleft; in the closed position, the glycine lid prevents ATP from leaving the cleft. Subsequent to phosphoryl transfer from the bound ATP to the bound peptide sequence, the glycine lid must rotate back to the open position before ADP can be released. Kinetic measurements show that the rate of ADP release is 20-fold slower than that of phosphoryl transfer, indicating the influence of the glycine lid on the rate of ki-nase reactions. Mutations in the glycine lid that inhibit its flexibility slow catalysis by protein kinase A even further.
Phosphoryl Transfer by Protein Kinases After substrates have bound and the catalytic core of protein kinase A has assumed the closed conformation, the phosphorylation of a serine or threonine residue on the target peptide can take place (Figure 3-18). As with all chemical reactions, phosphoryl transfer catalyzed by protein kinase A proceeds through a transition state in which the phosphate group to be transferred and the acceptor hydroxyl group are brought into close proximity. Binding and stabilization of the intermediates by protein kinase A reduce the activation energy of the phosphoryl transfer reaction, permitting it to take place at measurable rates under the mild conditions present within cells (see Figure 3-16). Formation of the products induces the enzyme to revert to its open conformational state, allowing ADP and the phosphorylated target peptide to diffuse from the active site.
Was this article helpful?
Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.