The Nature Of The Heme Environment Biology Essay


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Q1: List the residues that comprises the distal environment (and their type ). Comment on the nature of the heme environment.

The hemoglobin molecule is made up of four polypeptide chains: two alpha chains of 141 amino acid residues each and two beta chains of 146 amino acid residues each. The alpha and beta chains have different sequences of amino acids, but fold up to form similar three-dimensional structures. The four chains are held together by noncovalent interactions. There are four binding sites for oxygen on the hemoglobin molecule, because each chain contains one heme group. In the alpha chain, the 87th residue is histidine F8 and in the beta chain the 92nd residue is histidine F8. A heme group is attached to each of the four histidines. The heme consists of an organic part and an iron atom. The iron atom in heme binds to the four nitrogens in the center of the protoporphyrin ring. The hemoglobin molecule is nearly spherical, with a diameter of 55 angstroms. The four chains are packed together to form a tetramer. The heme groups are located in crevices near the exterior of the molecule, one in each subunit. Each alpha chain is in contact with both beta chains. However, there are few interactions between the two alpha chains or between the two beta chains.

      Each polypeptide chain is made up of eight or nine alpha-helical segments and an equal number of nonhelical ones placed at the corners between them and at the ends of the chain. The helices are named A-H, starting from the amino acid terminus, and the nonhelical segments that lie between the helices are named AB, BC, CD, etc. The nonhelical segments at the ends of the chain are called NA at the amino terminus and HC at the carboxyl terminus.

      To form the tetramer, each of the subunits is joined to its partner around a twofold symmetry axis, so that a rotation of 180 degrees brings one subunit into congruence with its partner. One pair of chains is then inverted and placed on top of the other pair so that the four chains lie at the corners of a tetrahedron. The four subunits are held together mainly by nonpolar interactions and hydrogen bonds. There are no covalent bonds between subunits. The twofold symmetry axis that relates the pairs of alpha and beta chains runs through a water-filled cavity at the center of the molecule. This cavity widens upon transition form the R structure to the T structure to form a receptor site for the allosteric effector DPG (2,3 diphosphoglycerate) between the two beta chains. The heme group is wedged into a pocket of the globin with its hydrocarbon side chains interior and its polar propionate side chains exterior.

      There are nine positions in the amino acid sequence that contain the same amino acid in all or nearly all species studied thus far. These conserved positions are especially important for the function of the hemoglobin molecule. Several of them, such as histidines F8 (His87) and E7 (His63), are directly involved in the oxygen-binding site. Phenylalanine CD1 (Phe43) and leucine F4 (Leu83) are also in direct contact with the heme group. Tyrosine HC2 (Tyr140) stabilizes the molecule by forming a hydrogen bond between the H and F helices. Glycine B6 (Gly25) is conserved because of its small size: a side chain larger than a hydrogen atom would not allow theB and E helices to approach each other as closely as they do. Proline C2 (Pro37) is important because it terminates the C helix. Threonine C4 (Thr39) and lysine H10 (Lys127) are also conserved residues, but their roles are uncertain.

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The haemoglobin  molecule consists of 4 polypeptide (globin) chains. In adults there are 2 alpha chains and 2 beta chains.

alpha chain  (141 amino acid residues):

val  -  leu ser pro ala asp lys thr asn val lys ala ala try gly lys val gly ala his ala gly glu tyr gly ala glu ala leu glu arg met phe leu ser phe pro thr thr lys thr tyr phe pro his phe  -  asp leu ser his gly ser ala  -   -   -   -   -  gln val lys gly his gly lys lys val ala asp ala leu thr asn ala val ala his val asp asp met pro asn ala leu ser ala leu ser asp leu his ala his lys leu arg val asp pro val asp phe lys leu leu ser his cys leu leu val thr leu ala ala his leu pro ala glu phe thr pro ala val his ala ser leu asp lys phe leu ala ser val ser thr val leu thr ser lys tyr arg

beta chain  (146 amino acid residues

val his leu thr pro glu glu lys ser ala val thr ala leu try gly lys val asn   -   -  val asp glu val gly gly glu ala leu gly arg leu leu val val tyr pro try thr gln arg phe phe glu ser phe gly asp leu ser thr pro asp ala val met gly asn pro lys val lys ala his gly lys lys val leu gly ala phe ser asp gly leu ala his leu asp asn leu lys gly thr phe ala thr leu ser glu leu his cys asp lys leu his val asp pro glu asn phe arg leu leu gly asn val leu val cys val leu ala his his phe gly lys glu phe thr pro pro val gln ala ala tyr gln lys val val ala gly val ala asp ala leu ala his lys tyr his

There are many similarities between the sequence of the alpha and beta chains, and - signs above hint at missing sections. Red text :This glutamic acid residue is replaced by valine in sickle cell anaemia (see later).

There is a peptide bond between each amino acid, so they are called residues because -H is removed from each intervening amino group, and -OH from the next -COOH group. At one end of each chain (the N terminal end) is an amino group, and at the other end (the C terminal end) is a carboxylic acid group. 

It is also very interesting to note similarities between the chains of haemoglobin and myoglobin, another protein which is involved in holding and passing on oxygen, but which consists of only one polypeptide chain (with 153 amino acid residues):

val leu ser glu gly glu trp gln leu val leu his val trp ala lys val glu ala asp val ala gly his gly gln asp ile leu ile arg leu phe lys ser his pro glu thr leu glu lys phe asp arg phe lys his leu lys thr glu ala glu met lys ala ser glu asp leu lys lys his gly val thr val leu thr ala leu gly ala ile leu lys lys lys gly his his glu ala glu leu lys pro leu ala gln ser his ala thr lys his lys ile pro ile lys tyr leu glu phe ile ser glu ala ile ile his val leu his ser arg his pro gly asn phe gly ala asp ala gln gly ala met asn lys ala leu glu leu phe arg lys asp ile ala ala lys tyr lys glu leu gly tyr gln gly

Haemoglobin (and myoglobin) produced in other organisms may have a slightly different amino acid sequence, and fewer or extra amino acids, but the next levels of structure are not greatly altered by these variations.

Q2: Which secondary structure element of the subunits make up the interface?

Hemoglobin is a globular protein with a heme group. In human adults humans the most common hemoglobin is a tetrameric structure that contains four subunits that includes two alpha polypeptides and two beta polypeptides that are non-covalently bound and hence denoted as a2b2.

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The heme group of hemoglobin consists of an iron atom that is present in a heterocyclic ring, called as a porphyrin. This iron atom present in the heme group of hemoglobin binds to the oxygen and is involved in oxygen transport.

Secondary structure refers to the hydrogen bonds present between the peptide groups in the main chain and it includes alpha helix and beta strands.

The protein part of the hemoglobin is known as"globin" Each alpha protein of hemoglobin consists of 141 amino acids and each beta protein consists of 146 amino acids. The alpha and beta globin proteins are similar in structure. The four polypeptides in hemoglobin secondary structure are bound to each other by hydrophobic interactions, salt bridges, and hydrogen bonds. Both alpha and beta globin proteins have similar secondary structure.

Hemoglobin secondary structure mainly consists of alpha helices and inter connected by short segments of non helical regions. Each alpha and beta globin proteins have eight helices. Hydrogen bond stabilizes the alpha helices. Beta strands are not present in the hemoglobin secondary structure and also hemoglobin secondary structure lack disulfide bonds. Each globin of hemoglobin is associated with a heme group. Intertwining sets of globin helices wrap tightly around heme group and gives rise to a compact tertiary structure

Q3: What are the distances between the iron atoms in the 4 subunits?

The distances between the iron atoms in the alpha and beta subunit in themselves are 36.42 A0 and distance between the iron atom in two different subunits are 24.26 A0.

Q4: What is the RMS co-ordinate difference in this region?

Analysis of the conformational differences between the oxy and deoxy forms of hemoglobin is complicated by shifting coordinate systems and correlated motions between different parts of the molecule. Methods independent of any frame of reference were used to study the differences in structure between the oxy and deoxy forms of the human hemoglobin αβ dimer. Differences between the deoxy and oxy dimer structures can be characterized as rearrangements of 15 substructures persisting between the two conformations. Such substructures are of two kinds, either rigid domains or tertiary substructures. Rigid domains are groups of residues for which all inter-residue distances are conformationally invariant. Residues belonging to a rigid domain do not have to be spatially contiguous nor must they have consecutive sequence numbers. The largest such substructure is a rigid core that spans both the α and β monomers and includes 44% of the dimer. Other rigid domains exist within the heme pockets.

An alternative but closely related view of the molecule is based on tertiary substructures. Unlike a rigid domain, a tertiary substructure must have consecutively numbered residues and the residue that ends one tertiary substructure begins the next. The decomposition of the dimer into tertiary substructures represents the dimer as a framework of connected stiff structural elements. Viewed as a set of tertiary substructures, the hemoglobin dimer has the same three principal functional elements: the dimer core and the α and β heme pockets, with the heme pockets held to the dimer core by CD and FG corners. The tertiary substructures that comprise the dimer core include 51% of the molecule. When ligands bind at the hemes, the FG corners communicate structural changes in the hemes to the dimer cores, which may mediate heme-heme cooperativity.

Q5: What is the behaviour of the iron atom? Which regions of backbone differ most?

In hemoglobin, each subunit contains a heme group, which is displayed using the ball-and-stick representation in. Each heme group contains an iron atom that is able to bind to one oxygen (O2) molecule. Because hemoglobin contains four heme groups, each hemoglobin protein can bind four oxygen molecules.

In the body, the iron in the heme is coordinated to the four nitrogen atoms of a porphyrin and also to a nitrogen atom of a histidine amino-acid residue in the hemoglobin protein. The sixth position (coordination site) around the iron of the heme is occupied by O2 when the hemoglobin protein is oxygenated.

Careful examination of demonstrates that the heme group is nonplanar when in its deoxygenated state; the iron atom is pulled out of the plane of the porphyrin toward the histidine residue to which it is attached. This nonplanar configuration is characteristic of the deoxygenated heme group and is commonly referred to as a "domed" shape. The valence electrons in the atoms surrounding the iron in the heme group and the valence electrons in the histidine residue form "clouds" of electron density. (Electron density refers to the probability of finding an electron in a region of space.) Because electrons repel one another, the regions occupied by the valence electrons in the heme group and in the histidine residue are pushed apart. Hence, the porphyrin adopts the domed nonplanar configuration in which the Fe is out of the plane of the porphyrin ring. However, when the heme group is in its oxygenated state, the porphyrin ring adopts a planar configuration in which the Fe lies in the plane of the porphyrin ring.

The planar and nonplanar configurations of the heme group have important implications for the rest of the hemoglobin protein. When the iron atom moves into the porphyrin plane upon oxygenation, the histidine residue to which the iron atom is attached is drawn closer to the heme group. This movement of the histidine residue shifts the position of other amino acids that are near the histidine. When the amino acids in the protein are shifted by the oxygenation of one of the heme groups, the structure of the interfaces between the four subunits is altered. This causes the whole protein to change its shape. In the new shape, it is easier for the other three heme groups to become oxygenated. Thus, the binding of one molecule of O2 to hemoglobin enhances the ability of hemoglobin to bind more O2 molecules. This property of hemoglobin is known as cooperative binding.

Q 6: The Heme , His F8 , the H-helix and the FG corner have been described as the allosteric core of hemeglobin (Gelin, Lee and Karplus, J. Mol. Biol., 171, 489 (1983). Why?

Exposure of crystals of Hb Cowtown in the T-state to oxygen pressures of up to about 40 torr at 15_C stabilises a fractional saturation of just over 0.4 without change of quaternary structure. Higher oxygen saturations produce a gradual, constant increase in fractional saturation, due apparently to an increase fraction of the molecules undergoing the T to R transition, even though there is no visible damage to the crystals. That transition prevented us from obtaining complete oxygen equilibrium curves in the T-state, a dif®culty that had also been encountered with crystals of des His 146b-Hb. To be sure that the fractional saturation really corresponds to the pure T-state, polarised absorption spectra were recorded for several hours on crystals ®rst equilibrated with humidi®ed helium and then exposed to de®ned oxygen pressures. In order to calculate oxygen equilibrium curves, we also needed to know the concentrations of metHb. They were calculated by tting the observed absorption spectra to linear combination of the spectra of pure T-state deoxyoxy- and metHb crystals. Reference spectra for fully deoxyHb and metHb Cowtown were obtained by bathing crystals in media containing either Na2S2O4 or ferricyanide (Figure 3a and c), but reference spectra for fully oxygenated crystals in the T-state were unobtainable due to the T to R transition. We therefore used as reference spectra those of fully oxygenated crystals in the R-state after exposure to oxygen at 740 torr at 10_C.

Despite the slight spectral differences between HbO2 in the T and R states, observed and calculated spectra ®tted well even for the highest

oxygen saturation (Figure 3d). Figure 4 shows the time-dependence of the fractional saturation with oxygen and of the fractional concentration of metHb. The rate of oxidation is seen to increase sharply with oxygen pressure. The oxygen equilibrium curves of the Hb Cowtown crystals obtained by ®tting the parameters of the Hill equation, p50 and n, to the observed points. p50 and n are 43.6(_1.9) torr and 0.99(_0.04) in light polarised along the a-axis and 44.7(_1.9) torr and 0.98(_0.04) in light polarised normal to the a-axis. This oxygen af®nity is about three times higher than that of Hb A crystals; similarly K1, the ®rst Adair (association) constant of Hb Cowtown, was found to be 1.3 to 1.8 times greater than that of HbA, depending on [Clÿ], at pH 7.4 and 25_C (Shih et al., 1984). Our results show that removal of the hydrogen bonds from His146b to Asp94b and Lys40a has an even greater effect on the oxygen af®nity of a crystal in which the molecules are clamped in the T-structure than in solution, where these constraints are absent, and they prove that the salt-bridges directly lower the oxygen af®nity of the T-structure. It has been argued that oxygen binding by crystals of Hb A might have been found to be noncooperative because some interaction between the a and b-haems was compensated by non-equivalence of their oxygen af®nities. However, the oxygen binding curves derived from analysis of the absorption spectra in light polarised parallel and perpendicular to the a-axis show no difference between the oxygen af®nities of the a and b-haems, so that haem-haem interaction in Hb Cowtown in the T-state must be absent. It was also found to be negligible in haemoglobin A encapsulated in silica gels (Shibayama & Saigo 1995; Bettati & Mozzarelli, 1997).

Q7: What are the major differences you observe from the alpha-1 subunit?

The alpha subunit of hemoglobin has several amino acid sequences that are conserved across many species and are essential to its function. The alpha subunit of hemoglobin is encoded by the 2 genes HBA1 and HBA2 both located on chromosome 16 (GeneCard, 2005). To determine which amino acid sequences are conserved, I compared the orthologs of HBA1 in Homo sapiens (humans) to 5 additional species including, Xenopus tropicalis (African clawed frog), Danio rerio (Zebra fish), Gallus gallus (Red jungle fowl), Mus musculus (mouse), and Rattus norvegicus (rat) using the Ensembl program. Figure 3 shows the 6 orthologs aligned and the important conserved regions highlighted. The stars indicate amino acids that are conserved between all of the species. As a general observation, the mouse ortholog of HBA is the most similar to human HBA, because it is the most evolutionarily related. The amino acid sequences that are conserved in all globin proteins (highlighted in blue) can be seen in Figure 3. There are also several conserved amino acids that are specifically important to HBA structure (highlighted in red) including: the phenylalanine (F) at position 44, which is in direct contact with the heme group; tyrosine (Y) at position 142, which stabilizes the hemoglobin molecule by forming hydrogen bonds between two of the helices; and glycine (G) at position 26, which is small and therefore allows two of the helices to approach each other, which is important to the structure of hemoglobin (Natzke, 1998). Additionally, there are several proteins found in the alpha subunit that are involved in the movement of the alpha and beta subunits (also highlighted in red) including: the tyrosine (Y) at position 43, which interacts with the beta subunit during the R state, and the arginine (N) at position 143, which interacts with the beta subunit during the T state (Gribaldo et al., 2003).

(shaded region are strural difference of alpha subunit)

The synthesis of haemoglobin A (HbA) is exquisitely coordinated during erythrocyte development to prevent damaging effects from individual α- and β-subunits1, 2. The α-haemoglobin-stabilizing protein (AHSP) binds α-haemoglobin (αHb), inhibits the ability of αHb to generate reactive oxygen species and prevents its precipitation on exposure to oxidant stress3, 4, 5. The structure of AHSP bound to ferrous αHb is thought to represent a transitional complex through which αHb is converted to a non-reactive, hexacoordinate ferric form5. Here we report the crystal structure of this ferric αHb-AHSP complex at 2.4 Šresolution. Our findings reveal a striking bis-histidyl configuration in which both the proximal and the distal histidines coordinate the haem iron atom. To attain this unusual conformation, segments of αHb undergo drastic structural rearrangements, including the repositioning of several α-helices. Moreover, conversion to the ferric bis-histidine configuration strongly and specifically inhibits redox chemistry catalysis and haem loss from αHb. The observed structural changes, which impair the chemical reactivity of haem iron, explain how AHSP stabilizes αHb and prevents its damaging effects in cells.

Q8: To what specific structural feature dose the term switch refer? Can you see it? How do the quaternary shifts effect specific interaction at the interface, how is this important in the co-operative mechanism?

The globin chain synthetic pattern and the extent of DNA methylation within embryonic, fetal, and adult beta-like globin gene domains were evaluated in greater than or equal to 90% purified human erythroblasts from yolk sacs and fetal livers in the 6- to 12-wk gestational period as well as from adult marrows. The 6-wk erythroblasts produce essentially embryonic epsilon chains, whereas the 12-wk erythroblasts synthesize largely fetal gamma globin and the adult marrow erythroblasts synthesize almost exclusively adult beta chains. In all phases of ontogenic development, a strong correlation exists between DNA hypomethylation in the close flanking sequences of globin genes and their expression. These results suggest that modulation of the methylation pattern may represent a key mechanism for regulating expression of human globin genes during embryonic leads to fetal and fetal leads to adult Hb switches in humans. In ontogenic development this mechanism might in turn correlate with a gradual modification of chromatin structure in the non-alpha gene cluster, thus leading to a 5' leads to 3' activation of globin genes in a balanced fashion.

The Fe-His stretching mode demonstrated that all of mutants Hbs take the T structure in the deoxy form under these experimental conditions. The UVRR change of the Trp residue of these mutants upon the T-R transition was the same as that in HbA, indicating that the T-R-dependent UVRR change of beta37Trp is not due to stacking with Tyr residues but is due to the formation or destruction of a hydrogen bond. The recombinant Hbs beta35Tyr --> Phe and beta35Tyr --> Thr both exhibited UVRR spectra identical with that of HbA, meaning that beta35Tyr is not responsible. In the spectra of des(beta146His,beta145Tyr)Hb with inositol hexaphosphate, the frequency shift of the Tyr RR bands was the same as that in HbA but the intensity enhancement in the CO form was small, suggesting that beta145Tyr contributes to a part of the intensity change, but scarcely relates to the frequency shift. In the spectra of Hb Rouen (alpha140Tyr --> His), the frequency shifts of bands at 1617 (Y8a) and 1177 (Y9a) cm-1 following ligation were half of those in HbA, while the intensity enhancement was not detected. This result means that alpha140Tyr is responsible for both the frequency shift and the intensity changes. It is suggested that the frequency shift of the Tyr RR bands upon the T --> R transition is due to changes in the hydrogen bonding state of alpha42- and alpha140Tyr and that the intensity enhancement is due to changes in the environment of the penultimate Tyr in both alpha and beta subunits (alpha140 and beta145). These alterations in the vibrational spectra clearly demonstrate which tyrosine residues are involved in the T-R transition as a result of modification of their local environments.

Co-operative interpreted their two-state model in terms of an equilibrium between two alternative structures, a tense one (T) with low oxygen affinity, constrained by salt-bridges between the C-termini of the four subunits, and a relaxed one (R) lacking these bridges. The equilibrium was thought to be governed primarily by the positions of the iron atoms relative to the porphyrin: out-of-plane in five-coordinated, high-spin deoxyhemoglobin, and in-plane in six-coordinated, low-spin oxyhemoglobin. The tension exercised by the salt-bridges in the T-structure was to be transmitted to the heme-linked histidines and to restrain the movement of the iron atoms into the porphyrin plane that is necessary for oxygen binding. At the beta-hemes, the distal valine and histidine block the oxygen-combining site in the T-structure; its tension was thought to strengthen that blockage. Finally, Perutz attributed the linearity of proton release with early oxygen uptake to the sequential rupture of salt-bridges in the T-structure and to the accompanying drop in pKa of the weak bases that form part of them.

Q9: What is the major effect upon going from T to R on the central cavity of the tetramer?

The relationship between the T, R, and R2 quaternary forms of hemoglobin is examined by computational experiments. Contrary to previous suggestions, we propose that the R quaternary form may lie on the pathway from T to R2. This proposal is consistent with four independent observations. (i) Difference distance maps are used to identify those parts of the molecule that undergo conformational change upon oxygenation. The simplest interpretation of these maps brackets R between T and R2. (ii) Linear interpolation from T to R2 passes through R. (iii) The well-known "switch" region (so called because, upon transition between the T and R quaternary forms, a residue from the beta 2 subunit toggles between two stable positions within the alpha 1 subunit) progresses from T through R to R2, successively. (iv) A hitherto-undocumented feature, diagnostic of the R structure, is noted within the alpha subunit: upon transformation from T to R, the beta-turns at the amino terminal of the E and F helices flip from one turn type to another. Upon transformation from R to R2, the latter turn--a strained conformation--flips back again.

Allosteric effects in hemoglobin arise from the equilibrium between at least two energetic states of the molecule: a tense state, T, and a relaxed state, R. The two states differ from each other in the number and energy of the interactions between hemoglobin subunits. In the T state, constraints between subunits oppose the structural changes resulting from ligand binding. In the R state, these constraints are released, thus enhancing ligand-binding affinity. In the present work, we report the presence of four sites in hemoglobin that are structurally stabilized in the R relative to the T state. These sites are Hisα103(G10) and Hisα122(H5) in each α subunit of hemoglobin. They are located at the α1β1 and α2β2 interfaces of the hemoglobin tetramer, where the histidine side chains form hydrogen bonds with specific residues from the β chains. We have measured the solvent exchange rates of side chain protons of Hisα103(G10) and Hisα122(H5) in both deoxygenated and ligated hemoglobin by NMR spectroscopy. The exchange rates were found to be higher in the deoxygenated-T than in ligated-R state. Analysis of exchange rates in terms of the local unfolding model revealed that the structural stabilization free energy at each of these two histidines is larger by ≈1.5 kcal/(mol tetramer) in the R relative to the T state. The location of these histidines at the intradimeric α1β1 and α2β2 interfaces also suggests a role for these interfaces in the allosteric equilibrium of hemoglobin.

Q10: What is an allosteric effector? Give examples and describe their principal binding sites.

Allosteric effect The binding of a ligand to one site on a protein molecule in such a way that the properties of another site on the same protein are affected. Some enzymes are allosteric proteins, and their activity is regulated through the binding of an effector to an allosteric site.

Regulatory effect that is transmitted over a distance within a protein. The binding of an effector in one site will change the catalytic behaviour of an enzyme or the binding affinity of a binding protein in a different part of the biomolecule. The classic example is haemoglobin where binding of oxygen to one of the four subunits increases the affinity of the others.

Allosteric effects occur because effector molecules are able to bring about conformational changes within the enzyme or protein. This may lead to disruption of the active site, the inability of the substrate molecule (the molecule undergoing change) to bind, or the inability of the products of the reaction to be released.

Allosteric enzymes (those subject to allosteric effects) usually consist of several polypeptide chains, associated together in a quaternary structure. They are large, complicated molecules, which usually exert their catalytic effect at a key point in a biochemical pathway.

There are three major groups which experience allosteric effects. In homotropic enzymes the substrate molecule can itself be an allosteric effector. In heterotropic enzymes there is a specific effector molecule, other than the substrate molecule of the reaction, and often the end product. Some multivalent regulatory enzymes can exhibit the two types at the same time.

An enzyme inhibitor is a molecule that binds to enzymes and decreases their activity. Since blocking an enzyme's activity can kill a pathogen or correct a metabolic imbalance, many drugs are enzyme inhibitors. They are also used as herbicides and pesticides. Not all molecules that bind to enzymes are inhibitors; enzyme activators bind to enzymes and increase their enzymatic activity.

The binding of an inhibitor can stop a substrate from entering the enzyme's active site and/or hinder the enzyme from catalysing its reaction. Inhibitor binding is either reversible or irreversible. Irreversible inhibitors usually react with the enzyme and change it chemically. These inhibitors modify key amino acid residues needed for enzymatic activity. In contrast, reversible inhibitors bind non-covalently and different types of inhibition are produced depending on whether these inhibitors bind the enzyme, the enzyme-substrate complex, or both.

Many drug molecules are enzyme inhibitors, so their discovery and improvement is an active area of research in biochemistry and pharmacology. A medicinal enzyme inhibitor is often judged by its specificity (its lack of binding to other proteins) and its potency (its dissociation constant, which indicates the concentration needed to inhibit the enzyme). A high specificity and potency ensure that a drug will have few side effects and thus low toxicity.

Enzyme inhibitors also occur naturally and are involved in the regulation of metabolism. For example, enzymes in a metabolic pathway can be inhibited by downstream products. This type of negative feedback slows flux through a pathway when the products begin to build up and is an important way to maintain homeostasis in a cell. Other cellular enzyme inhibitors are proteins that specifically bind to and inhibit an enzyme target. This can help control enzymes that may be damaging to a cell, such as proteases or nucleases; a well-characterised example is the ribonuclease inhibitor, which binds to ribonucleases in one of the tightest known protein-protein interactions.[1] Natural enzyme inhibitors can also be poisons and are used as defences against predators or as ways of killing prey.

For many years seven transmembrane domain G protein-coupled receptors (GPCRs) were thought to exist and function exclusively as monomeric units. However, evidence both from native cells and heterologous expression systems has demonstrated that GPCRs can both traffic and signal within higher-order complexes. As for other protein-protein interactions, conformational changes in one polypeptide, including those resulting from binding of pharmacological ligands, have the capacity to alter the conformation and therefore the response of the interacting protein(s), a process known as allosterism. For GPCRs, allosterism across homo- or heteromers, whether dimers or higher-order oligomers, represents an additional topographical landscape that must now be considered pharmacologically. Such effects may offer the opportunity for novel therapeutic approaches. Allosterism at GPCR heteromers is particularly exciting in that it offers additional scope to provide receptor subtype selectivity and tissue specificity as well as fine-tuning of receptor signal strength. Herein, we introduce the concept of allosterism at both GPCR homomers and heteromers and discuss the various questions that must be addressed before significant advances can be made in drug discovery at these GPCR complexes.

Cell-surface receptors are the targets for more than 60% of current drugs. Traditionally, optimizing the interaction of lead molecules with the binding site for the endogenous agonist (orthosteric site) has been viewed as the best means of achieving selectivity of action. However, recent developments have highlighted the fact that drugs can interact with binding sites on the receptor molecule that are distinct from the orthosteric site, known as allosteric sites. Allosteric modulators could offer several advantages over orthosteric ligands, including greater selectivity and saturability of their effect.