19 March 2007
Biol 6312
The Structural Basis of Protein Function
Function can be explained on several levels: The function of a kinase enzyme is phosphotransferase activity, but on a higher level it might be part of signal transduction system. (Fig. 2-1)
Also, some proteins have several functions, in some cases, both well-known functions and newly-discovered functions.
The four fundamental functions of proteins:
Recognition, Complementarity and Active Sites
Binding is important for proteins involved in signaling, catalysis or transport.
1. The molecule that is bound, the ligand, can be small or large (another protein).
2. Binding is non-covalent, and uses the standard interactions
3. Specificity is usually high (the affinity is high relative to that of similar molecules)
4. Specificity is a consequence of complementarity of shape and polarity
A protein can form a binding pocket for a ligand that is quite different from that found on the surface, or in water itself.
Such specialized environments are particularly important for enzymes. For example, enzymes often have groups with shifted pKa values to facilitate catalysis. We are familiar with groups of opposite charge forming an ion pair, which might contribute to the stability of a protein.
1. In an enzyme two groups that could ionize might be found in a nonaqueous environment, and held too far apart to transfer the proton, leaving both in an un-ionized state. That way each might participate in catalysis, one as a strong acid, and one as a strong base. This could not generally occur in solution.
2. The environment of a single group can cause a shift in its pKa. Two lysine residues that are close to each other, will make it more difficult to protonate the second one. (Fig. 2-3) (It becomes a stronger acid, its proton affinity down.) This can facilitate its role in proton transfer. Likewise for a lysine found in a nonpolar environment, this will favor the uncharged, unprotonated state. This can facilitate some chemcial steps, such as a nucleophilic attack.
Flexibility and Protein Function
Proteins are commonly thought of a "molecular machines", but in fact they are generally much more flexible than macroscopic machines made of metal. The early metaphor of Emil Fischer of the "lock and key", for an enzyme and its substrate has some validity, but is not entirely accurate. Most enzymes are highly specific for their substrates, while there are some exceptions. However, many enzymes change conformation after binding one of their substrates. This is known as induced-fit, as we have seen previously.
(Fig. 2-4) Tight fit between enzyme and substrate
Proteins are inherently flexible because their conformations are maintained by numerous weak interactions. At any moment many of them might be broken, leading to local, transient rearrangements. This flexibility can allow an enzyme to bind a variety of slightly different substrates, but it also allows a variety of different inhibitors to bind to a single enzyme. See the example of the HIV-protease. (Fig. 2-5)
Binding of ligands can range from loose binding (KD = 10-3 M) to extremely tight binding (KD = 10-12 M) Tight binding is usually associated with subtle conformational changes that allow greater surface complementarity.
One property of enzymes that function at very high temperatures in thermophilic organisms is greater rigidity. (Fig. 2-6) This can be seen as shorter N- and C-termini, and shorter loops, in some cases.
The flexibility of proteins varies greatly. Some proteins undergo large conformational changes, for example, the binding of AMP by adenylate kinase. (Fig. 2-7)
Location of Binding Sites
Binding sites vary in size and in other characteristics, depending on whether the partner is a small molecule, another protein, or a nucleic acid. Protein-protein interaction surfaces can be quite large, hundreds of square Å, and can be discontinuous surfaces, (Fig. 2-8). (Fig. 2-9)
Small ligands often bind in cavities. This allows special environments that might be necessary for tight binding. Protein flexibility is often necessary to allow the ligand to enter the cavity. (Fig. 2-10)
We have previously discussed the location of active sites in several standard folds. From another point of view, the binding of ligands or substrates often occurs at the interface of two domains, or of two subunits. (Fig. 2-11)
For example, the binding of ATP in the ATP synthase occurs at the switch region near the C-terminal ends of the parallel β-sheet (Rossmann fold), but it also occurs at the interface of two subunits.
Nature of Binding Sites
Prediction Server Q-site Finder
Laurie AT, Jackson RM.
Q-SiteFinder: an energy-based method for the prediction of protein-ligand binding sites.
Bioinformatics. 2005 21:1908-16
One common feature of a ligand-binding site is hydrophobic surface. Due to the hydrophobic effect, the binding of a ligand is more favorable if a larger amount of hydrophobic surface is buried upon binding.
Hydrophobic surfaces on proteins can lead to non-specific associations between proteins, otherwise known as aggregation, which is dangerous. Binding sites for small ligands are usually concave, with the hydrophobic surface less accessible. (Fig. 2-12)
Many protein-protein interactions are transient, which requires that they have only moderate stability. Relatively small hydrophobic patches will usually lead to moderate affinity. Transient binding is important in signal transduction pathways. More stable complexes can arise when a complex of two proteins creates a new binding site for a third protein. The 3-subunit complex could be more stable than any combination of 2. Covalent modification, such as phosphorylation, can alter conformation, and weaken binding, allowing proteins to dissociate and find new partners. (Fig. 2-13) This is referred to as partner swapping.
Domain swapping allows more stable complexes to form. As illustrated in (Fig. 2-14), in a trimeric complex, part of one subunit invades another and displaces the corresponding part. The domain-swapped complexes can be transient or long-lived. (Jmol)
Proteins should generally be considered to have a layer of water molecules bound to their surfaces. This can be demonstrated by ultracentrifugation, because the water contributes mass. As we have seen, the water molecules will appear in the crystal structures. (Jmol). Therefore, when a ligand binds to a protein, it must not only be desolvated (stripped of water), but must also displace the waters residing at or near the binding site. The energetics of this process can be complicated.
Distinguishing between interactions that contribute to specificity and to affinity, is based on whether the interactions are directional or not. Hydrogen bonding is directional, so it usually contributes to specificity. Nonpolar interactions are less directional, and therefore they will contribute to affinity, but not specificity. In the case of a fatty acid binding protein, specificity is with respect to the carboxyl group of the fatty acid. (Fig. 2-15) (Jmol)
Structural Proteins
Cells are highly organized, largely due to structural proteins inside.
Well-known structural proteins include silk, collagen, keratin, elastin.
They can be held together by numerous weak, noncovalent interactions (the usual H-bonding, ion pairing, and hydrophobic), and also by covalent linkages (disulfides, or other specific, enzyme-catalyzed bonds). (Fig. 2-17)
Some structural proteins are also enzymes. Muscle is such an example in which ATPase enzymes are coupled to the filaments.
Some structural proteins function as scaffolds, for the binding of numerous other proteins. Ste5p forms a scaffold for the MAP kinase signalling pathway of response to mating type pheromones in yeast. It not only binds 3 kinases, which function sequentially, but also binds a β subunit of a heterotrimeric G protein. (Fig. 2-18)
Overview of Catalysis
Catalysts accelerate the rate of a reaction without undergoing any permanent change.
Most biological catalysts are proteins, and are called enzymes. RNA molecules can also be catalysts, such as the large subunit of the ribosome, and are termed ribozymes.
The catalysis of chemical reactions by enzymes can be extremely rapid, if one compares those rates to uncatalyzed rates. The decarboxylation of orotidine 5'-monophosphate to uridine 5'-monophosphate is extremely slow, but is enhanced by 17 orders of magnitude by the enzyme orotidine 5'-monophosphate decarboxylase (ODCase). (Fig. 2-19)
Other enzymes only accelerate reactions by a few orders of magnitude, shown Fig. 2-20.
Remember that chemical reactions, or transformations, can be thought of in terms of an equilibrium. The associated equilibrium constant can be described by the concentration of reactants and products at equilibrium, or by the ratio of the forward and reverse rate constants. A catalyst does not affect the concentrations at equilibrium. It does increase the forward rate constant, but it affects the reverse rate by the same factor. And so, the ratio (the equilibrium constant) is not changed. By increasing the forward rate constant, a catalyst increases the rate of product formation, and shortens the time to reach equilibrium.
Enzymes generally use multiple different ways to catalyze reactions. There is not a single explanation for the power of enzymatic catalysis. We will explore some of the different means of catalysis. They include proximity and orientation of substrates with catalytic groups of the enzyme. Microenvironments at the binding sites can also enhance the catalytic properties of amino acid side chains and of co-factors.
Transition state theory is often used to analyze enzyme-catalyzed reactions. The reaction proceeds through a high energy state, or barrier, that is called the transition state. The energy required to overcome this barrier is the activation energy. The transition state is the structure of the highest free energy. Catalysis requires that the free energy barrier be reduced. This can be accomplished by simply stabilizing the transition state, or the enzyme can cause the reaction to take a different path, perhaps with several intermediates along the way, resulting in several lower barriers. (Fig. 2-21)
Active-Site Geometry
In general, substrates must find the active-site of an enzyme by random collisions. For charged substrates, there can be an attractive potential around the enzyme, that allows the substrate to be pulled towards the active-site. An example is Cu-Zn superoxide dismutase (Fig. 2-22).
Electrostatic interactions can also help to orient the substrate in the active-site (Fig. 2-23)
Once at the active-site, a variety of groups from the enzyme will help to orient the substrate precisely, such that the catalytic groups are all properly placed. Co-factors may be involved in the catalysis also. (Fig. 2-24).
Sometimes the groups of the enzyme that interact with the substrate can be divided into those that contribute to specificity and those that contribute to reactivity. In that case, it might be possible to change the specificity of the enzyme without affecting its reactivity, through site-directed mutagenesis.
The Proximity Factor
Some enzymes promote catalysis primarily through simple proximity effects. In other words, it may be sufficient to simply bring the two substrates together, in the proper orientation, and at a close distance, to cause a reaction. This is called the proximity factor. An example is aspartate transcarbamoylase (ATCase), which is an important enzyme in pyrimidine biosynthesis.
carbamoyl-phosphate + aspartate → N-carbamoyl-aspartate + phosphate
A co-crystal of the enzyme with the inhibitor PALA (N-phosphoacetyl-L-aspartate) shows that no catalytic groups appear to be near the substrates. (Fig. 2-25). The reactivity comes from the substrates themselves, as they are held close together.
Ground-State Destabilization
The enzyme chorismate mutase appears to destabilize its substrate chorismate, by forcing it into an unusual "chair" conformation. This promotes catalysis because the conformation is on the path to the product. That is, it resembles the transition-state. (Fig. 2-26).
Stabilization of Transition States
Enzymes are often complementary to the transition state of the reaction they catalyze. That includes both shape complementarity and charge complementarity. This reduces the free energy of the transition state, or lowers the barrier. (Fig. 2-27) The enzyme might also stabilize the substrate, i.e. bind it tightly. But, in order to be a catalyst, the enzyme must stabilize the transition state more than it does the substrate.
In the example of citrate synthase, the enzyme stabilizes the charge distribution that must develop during the reaction:
oxaloacetate + acetyCoA → citrate + CoA
Two carbonyl groups must become partially negative, and these are stabilized by positively-charged Histidine residues at the active site of citrate synthase (Fig. 2-28)
The complementarity necessary for transition state stabilization might require a conformational change in the enzyme, such as in Phosphoglycerate kinase (Fig. 2-29) You can see the change in secondary structure of a loop on the left, and the binding of an essential Magnesium ion (green).
Exclusion of Water
Some enzyme use the exclusion of water from the active site to improve catalysis. This is true for enzymes that transfer Hydride ions (H-) from NADH. This is important since water would tend to prevent such a transfer. It is accomplished by having a loop of the protein fold over the substrates after they are bound. (Fig. 2-30) (Jmol) The example shown is lactate dehydrogenase.
lactate + NAD → pyruvate + NADH
Although the loops must open and close each time the enzyme turns over, the actual rate of motion is not fast since the distances (10 Å) are so small.
Redox Reactions
First of the four common types of reactions (Fig. 2-31)
They include oxidation of Carbons, from CH to COH, or COH to C=O, and desaturation of C-C bonds.
They typically require cofactors such as NADH, NADPH, flavins FAD or FMN, or metals.
Addition/Elimination Reactions
Second of the four common types of reactions
Examples include the addition of water to the double bond of fumarate (Fumarase), and the addition of acetate to the carbonyl carbon of oxaloacetate (Citrate synthase). (Fig. 2-32)
Hydrolysis Reactions
Third of the common types of reactions
Examples include hydrolysis of proteins (Trypsin) and nucleic acids (Ribonuclease). These are favorable reactions, and they can be reversed to make proteins or nucleic acids, if the substrates are activated first through phospho-esters. (Fig. 2-33)
Decarboxylation Reactions
Fourth of the common types of reactions.
The removal of a Carbon atom is difficult, so to shorten a carbon chain, it usually proceeds through a decarboxylation. So, the last Carbon is oxidized to a carboxyl group, and then removed. Examples include pyruvate decarboxylase, which converts pyruvate to acetaldehyde + carbon dioxide. The stability of carbon dioxide makes these reactions more favorable. (Fig. 2-34)
Active-Site Chemistry: Acid-base chemistry
The transfer of a proton from the substrate to the enzyme, or from the enzyme to the substrate, is the most common element of enzyme catalysis.
Protons are transfered from groups of low affinity (acids) to groups of high affinity (bases). The affinity is measured by the pKa value, which is the pH at which 50% of a group is protonated.
Strong acids have pKa values less than 2, while strong bases have pKa values greater than 12. Strong acids are essentially totally dissociated at pH 7, while strong bases are toatlly protonated at pH 7.
Weak acids have pKa values between 4 and 7, while weak bases have pKa values between 7 and 10. They are all partially dissociated at pH 7. Examples are shown in Fig. 2-35.
In protein environments the pKa values can be shifted by several units from the standard values. A carboxyl group, such as the side chain of glutamate, can have its pKa value shifted up (making it a weaker acid) by a nonpolar environment. The charged form (negative here) is destabilized by a nonpolar environment, relative to an aqueous environment. That shifts the pKa up, since it will take a higher pH to deprotonate the group. This occurs in the enzyme lysozyme, which utilizes a protonated glutamic acid at its active site. (Fig. 2-36)
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Copyright 2007, Steven B. Vik, Southern Methodist University