H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 32
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This hypothetical reaction pathwaydepicts the changes in free energy G as a reaction proceeds. Areaction will take place spontaneously only if the total G of theproducts is less than that of the reactants (G). However, allchemical reactions proceed through one or more high-energytransition states, and the rate of a reaction is inverselyproportional to the activation energy (G‡), which is thedifference in free energy between the reactants and the highestpoint along the pathway. Enzymes and other catalysts acceleratethe rate of a reaction by reducing the free energy of thetransition state and thus G‡.production of energy by the conversion of glucose and oxygen into carbon dioxide and water. Other enzymes are present only in a particular type of cell because they catalyzechemical reactions unique to that cell type (e.g., the enzymesthat convert tyrosine into dopamine, a neurotransmitter, innerve cells).
Although most enzymes are located within cells,some are secreted and function in extracellular sites such asthe blood, the lumen of the digestive tract, or even outsidethe organism.The catalytic activity of some enzymes is critical to cellular processes other than the synthesis or degradation of molecules. For instance, many regulatory proteins and intracellularsignaling proteins catalyze the phosphorylation of proteins,and some transport proteins catalyze the hydrolysis of ATPcoupled to the movement of molecules across membranes.An Enzyme’s Active Site Binds Substratesand Carries Out CatalysisCertain amino acid side chains of an enzyme are importantin determining its specificity and catalytic power.
In the native conformation of an enzyme, these side chains arebrought into proximity, forming the active site. Active sitesthus consist of two functionally important regions: one thatrecognizes and binds the substrate (or substrates) and another that catalyzes the reaction after the substrate has been75bound. In some enzymes, the catalytic region is part of thesubstrate-binding region; in others, the two regions are structurally as well as functionally distinct.To illustrate how the active site binds a specific substrateand 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 cellularproteins, often in response to external signals.
Because theeukaryotic protein kinases belong to a common superfamily, the structure of the active site and mechanism of phosphorylation are very similar in all of them. Thus proteinkinase A can serve as a general model for this important classof enzymes.The active site of protein kinase A is located in the 240residue “kinase core” of the catalytic subunit. The kinasecore, which is largely conserved in all protein kinases, is responsible for the binding of substrates (ATP and a target peptide sequence) and the subsequent transfer of a phosphategroup from ATP to a serine, threonine, or tyrosine residuein the target sequence. The kinase core consists of a large domain and small one, with an intervening deep cleft; the activesite comprises residues located in both domains.Substrate Binding by Protein Kinases The structure of theATP-binding site in the catalytic kinase core complements thestructure of the nucleotide substrate.
The adenine ring of ATPsits snugly at the base of the cleft between the large and thesmall domains. A highly conserved sequence, Gly-X-Gly-XX-Gly-X-Val (X can be any amino acid), dubbed the “glycinelid,” closes over the adenine ring and holds it in position (Figure 3-17a). Other conserved residues in the binding pocketstabilize the highly charged phosphate groups.Although ATP is a common substrate for all protein kinases, the sequence of the target peptide varies among different kinases. The peptide sequence recognized by proteinkinase A is Arg-Arg-X-Ser-Y, where X is any amino acid andY is a hydrophobic amino acid.
The part of the polypeptidechain containing the target serine or threonine residue isbound to a shallow groove in the large domain of the kinasecore. The peptide specificity of protein kinase A is conferredby several glutamic acid residues in the large domain, whichform salt bridges with the two arginine residues in the target peptide. Different residues determine the specificity ofother 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 areseparated enough that substrate molecules can enter andbind.
When the active site is occupied by substrate, the domains move together into the closed position. This change intertiary structure, an example of induced fit, brings the target peptide sequence sufficiently close to accept a phosphate76CHAPTER 3 • Protein Structure and Function(a)Glycine lidSmall domainTargetpeptideNucleotidebindingpocketLarge domain(b)Glycine lidSmalldomainActive siteLargedomainthe active site. In the open position, ATP can enter and bindthe active site cleft; in the closed position, the glycine lid prevents ATP from leaving the cleft.
Subsequent to phosphoryltransfer from the bound ATP to the bound peptide sequence,the glycine lid must rotate back to the open position beforeADP can be released. Kinetic measurements show that the rateof ADP release is 20-fold slower than that of phosphoryl transfer, indicating the influence of the glycine lid on the rate of kinase reactions. Mutations in the glycine lid that inhibit itsflexibility slow catalysis by protein kinase A even further.Phosphoryl Transfer by Protein Kinases After substrates havebound and the catalytic core of protein kinase A has assumedthe closed conformation, the phosphorylation of a serine orthreonine residue on the target peptide can take place (Figure3-18).
As with all chemical reactions, phosphoryl transfer catalyzed by protein kinase A proceeds through a transition statein 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 Areduce the activation energy of the phosphoryl transfer reaction, permitting it to take place at measurable rates under themild conditions present within cells (see Figure 3-16). Formation of the products induces the enzyme to revert to its openconformational state, allowing ADP and the phosphorylatedtarget peptide to diffuse from the active site.Vmax and Km Characterize an Enzymatic ReactionOpenClosed▲ FIGURE 3-17 Protein kinase A and conformationalchange induced by substrate binding.
(a) Model of thecatalytic subunit of protein kinase A with bound substrates; theconserved kinase core is indicated as a molecular surface. Anoverhanging glycine-rich sequence (blue) traps ATP (green) in adeep cleft between the large and small domains of the core.Residues in the large domain bind the target peptide (red).
Thestructure of the kinase core is largely conserved in othereukaryotic protein kinases. (b) Schematic diagrams of open andclosed conformations of the kinase core. In the absence ofsubstrate, the kinase core is in the open conformation. Substratebinding causes a rotation of the large and small domains thatbrings the ATP- and peptide-binding sites closer together andcauses the glycine lid to move over the adenine residue of ATP,thereby trapping the nucleotide in the binding cleft. The model inpart (a) is in the closed conformation.group from the bound ATP. After the phosphorylation reaction has been completed, the presence of the products causesthe domains to rotate to the open position, from which theproducts are released.The rotation from the open to the closed position alsocauses movement of the glycine lid over the ATP-binding cleft.The glycine lid controls the entry of ATP and release of ADP atThe catalytic action of an enzyme on a given substrate can bedescribed by two parameters: Vmax, the maximal velocity ofthe reaction at saturating substrate concentrations, and Km(the Michaelis constant), a measure of the affinity of an enzyme for its substrate (Figure 3-19).
The Km is defined as thesubstrate concentration that yields a half-maximal reaction1rate (i.e., 2 Vmax). The smaller the value of Km, the moreavidly an enzyme can bind substrate from a dilute solutionand the smaller the substrate concentration needed to reachhalf-maximal velocity.The concentrations of the various small molecules in acell vary widely, as do the Km values for the different enzymes that act on them. Generally, the intracellular concentration of a substrate is approximately the same as or greaterthan the Km value of the enzyme to which it binds.Enzymes in a Common Pathway Are OftenPhysically Associated with One AnotherEnzymes taking part in a common metabolic process (e.g.,the degradation of glucose to pyruvate) are generally locatedin the same cellular compartment (e.g., in the cytosol, at amembrane, within a particular organelle).
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