Enzymes That Bind Nucleotides Nucleotides play a central role in cellular metabolism, in which they are the major currency of energy exchange. They channel the energy released during the catabolism of food or captured from light during photosynthesis into the energy-requiring processes of the organism, such as synthesis of DNA, RNA, and proteins, active transport across membranes, and movement. Chemically, a nucleotide consists of a base covalently linked to a sugar, which, in turn, is linked to at least one phosphate group (Figure 10.1a). Energytransfer processes involving nucleotides can be divided into two distinct classes depending on which parts of the nucleotide engage in the energy transfer. The most familiar class involves high-energy phosphate bonds in triphosphates, such as ATP or GTP. The energy released by hydrolysis of these high-energy phosphate bonds is used to drive a large variety of metabolic processes. We will briefly discuss some kinases where phosphoryl groups are transferred between such nucleotides and metabolites. The second class utilizes the base for electron transfer through hydrogen atoms in oxidation-reduction (redox) processes. The usual bases, A,T,G,C,U, that are present in RNA and DNA are not suitable for this purpose. Instead, two different bases are used, nicotinamide (Figure 10.1) and isoalloxazine (flavin) ( Figure 10.2). Humans are unable to synthesize these molecules, which therefore have to be provided in the diet as vitamins. In nucleotides these two bases Figure 10.1 (a) The basic unit of cofactors involved in energy transfer is a nucleotide that is built up from a base that is linked to a ribose sugar, a nucleoside, that in turn is linked to a phosphate group. There are two classes of energy-transfer mechanisms: one uses energy of phosphate hydrolysis; the other uses electron transfer through hydrogen atoms on the base. (b) Nicotinamide adenine dinucleotides, NAD and NADP, are used as cofactors in a variety of enzymes, frequently dehydrogenases, to shuttle electrons between different oxidation-reduction systems. NAD contains an adenine-ribose-phosphate AMP part coupled through a pyrophosphate bond to a nicotinamide-ribose-phosphate NMN part. NADP has an extra phosphate linked to the 2'-OH ribose group of the AMP part. The oxidized forms, NAD and NADP, accept electrons in the form of a hydride ion that binds covalently to the C4 atom of the nicotinamide moiety, giving the reduced forms NADH and NADPH. (c) These cofactors bind to dehydrogenases in an energetically unfavorable form, shaped like a boomerang, that is stabilized by interactions with the protein.
Figure 10.2 Certain redox-active enzymes, such as oxidases, use flavin nucleotides, FMN and FAD, as cofactors. The electron acceptor in these cofactors is a fused three-membered ring system, isoalloxazine, which is linked to a sugar, ribitol. The FMN cofactor is a mononucleotide where the riboflavin moiety (nucleoside) is coupled to a phosphate group at the end of the ribitol chain. This FMN moiety is coupled to an adenosine-monophosphate, AMP nucleotide, to form a complete FAD molecule. The reduced forms of the cofactors have accepted two hydrogen atoms (red) in their isoalloxazine ring system. can be reduced by accepting hydrogen atoms at specific positions. The transfer of hydrogen from a substrate to the nucleotide base is catalyzed by the dehydrogenases and oxidases. The electron transfer and the high-energy phosphate systems are coupled in the mitochondria through the process of oxidative phosphorylation, where the energy from such reduced nucleotides drives the synthesis of ATP. Nicotinamide is present in the dinucleotide NAD (nicotinamide adenine dinucleotide) as well as in NADP, which has an extra phosphate group (Figure 10.1). An NAD molecule comprises adenine-ribose-phosphate-phosphate-ribose-nicotinamide. The first half, AMP, is linked to the second half, NMN (nicotinamide mononucleotide), through a pyrophosphate bond. The flavin base is present in two redox-active nucleotides, FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide) (Figure 10.2). FMN comprises a flavin base linked to the linear sugar alcohol ribitol (instead of ribose), which is linked to a phosphate. The dinucleotide FAD consists of AMP linked to FMN by a pyrophosphate bond. These redox-active nucleotides are called coenzymes in contrast to ATP, which is also required for the activity of many enzymes but is not referred to as a coenzyme. This distinction is mainly semantic and has been kept for historical reasons. The fundamental role of nucleotides in metabolism implies that nucleotide-binding enzymes must have arisen very early in the evolution of living organisms. This then raises the question of whether the multitude of different nucleotide-binding enzymes that exist today evolved independently of each other, or are they rather the descendants of one or a small number of primitive, ancestral nucleotide-binding proteins? What do we know about how enzymes bind redox-active nucleotide coenzymes, and does either the primary structure or the three-dimensional structure of these enzymes tell us anything about their evolution? The structures of several NAD-dependent dehydrogenases are known The NAD-dependent dehydrogenases comprise one of the largest and beststudied families of nucleotide-binding proteins: over 100 different members have already been identified. We will discuss the three-dimensional structures of four of these enzymes, those that catalyze oxidation of alcohol (LADH),
lactate (LDH), malate (MDH), and glyceraldehyde-3-phosphate (GAPDH) (see Table 10.1). Table 10.1 NAD-dependent dehydrogenases of known structure Enzyme Source Group Resolution (A) Year Alcohol dehydrogenase (LADH) horse liver Carl Bränden 2.4 1973 Lactate dehydrogenase (LDH) Uppsala, Sweden dogfish Michael Rossmann 2.0 1970 Malate dehydrogenase (MDH) pig heart Purdue University Len Banaszak 3.0 1972 Glyceraldehyde-3-phosphate lobster St Louis Michael Rossmann 2.9 1973 dehydrogenase (GAPDH) Purdue University Glyceraldehyde-3-phosphate B. stearothennophilous Alan Wonacott dehydrogenase (GAPDH) London 2.2 1977 The chemical reactions catalyzed by these four enzymes are in principle very simple. An alcohol group of the substrate is oxidized by the transfer of a hydrogen atom from the carbon atom that binds the hydroxyl group to the oxidized form of the coenzyme, NAD (Figure 10.3). In addition, a proton is removed from the alcohol hydroxyl group. For GAPDH there is an additional phosphorylation reaction coupled to the dehydrogenation step. All enzymatic reactions are reversible and in principle can go in both directions. Alcohol dehydrogenases from mammals and yeast provide illuminating examples of this principle. These enzymes have homologous amino acid sequences and catalyze the same reactions using the same mechanism. In mammals, ingested ethanol is removed from the body by oxidation to acetaldehyde, which is further oxidized to acetic acid in a subsequent enzymatic reaction. Certain individuals have a low alcohol tolerance due to mutations in these enzymes. In yeast, on the other hand, ethanol is produced from acetaldehyde during anaerobic fermentation of sugar molecules. Due to this subtle in vivo difference in the ability to influence the levels of ethanol and acetaldehyde, we, on the one hand, can produce alcohol-containing beverages in breweries and wineries and, on the other hand, can consume these drinks without toxic effects at moderate concentrations. During dehydrogenation the hydrogen atom is directly transferred from the substrate to the coenzyme. These enzymes, therefore, not only must recognize and bind both NAD and the correct substrate, but also must position them in the active site sufficiently close and in the correct orientation to allow direct hydrogen transfer to take place (Figure 10.4). All the NAD-dependent dehydrogenases use the same coenzyme, NAD, for similar chemical reactions, but the substrates differ in their size and shape and in the charge of the groups attached to the reactive alcohol. Are these functional similarities and differences reflected in the structures of these enzymes as we might expect them to be? The dehydrogenase polypeptide chains are modular Dehydrogenases are in general fairly large protein molecules; LDH and GAPDH are tetramers of four identical polypeptide chains, whereas MDH and LADH are dimers. The lengths of their polypeptide chains vary slightly, but they are all around 350 residues and the amino acid sequences are known. There is significant sequence homology between LDH and MDH; this is reflected in the three-dimensional structures of their subunits, which are essentially the same. Therefore, we will only discuss in detail one of these enzymes, LDH. No significant sequence homology can be detected, however, between LDH, GAPDH, and LADH, not even in small local regions of the polypeptide chains. From sequence comparisons alone one would, therefore, conclude that they are completely unrelated enzymes. But the three-dimensional structures tell a different story. Figure 10.3 The chemical reactions catalyzed by the dehydrogenases LADH, LDH, MDH, and GAPDH are in principle quite simple. During oxidation a hydride ion (red) is transferred from the substrate to NAD. In addition a proton (blue) is released from the alcohol group of the substrate to the solvent. Different dehydrogenases have different specificities for the side chains R 1 and R 2 that give the enzymes their substrate specificities.
The long polypeptide chains of these four dehydrogenases fold into two clearly separated domains. This is illustrated by the structural diagrams of LADH and GAPDH in Figure 10.5. The two domains have different functions, and each domain is therefore a functional module. One of them binds the coenzyme, NAD, and the second binds the substrate and provides the amino acids that are necessary for catalysis. We have discussed the concept that proteins are constructed in a modular fashion, with separate domains along the polypeptide chain fulfilling different functions, in previous chapters, but historically the structures of the dehydrogenases provided one of the first clear-cut examples of this principle. In LDH and GAPDH the NAD-binding domains are formed from the N- terminal portion of the polypeptide chains, whereas this domain is formed by the C-terminal region of LADH (Figure 10.6). In other words, the functionally similar NAD-binding domains occur in different regions of the polypeptide chain in these dehydrogenases. This is reminiscent of the situation with the DNA-binding domains in CAP and lambda repressers that are respectively C terminal and N terminal. The active site of these enzymes is in a cleft between the two domains. The substrate and the NAD binding sites in their respective domains are oriented so that the reactive part of the bound coenzyme, C4 of the nicotinamide ring, is in close proximity to the hydrogen atom to be transferred from the substrate. In LADH and GAPDH the domains are flexible and move closer to each other during catalysis so that the reactants are completely shielded from the solvent during hydrogen transfer. Figure 10.4 The nicotinamide ring of the coenzyme NAD and the substrate, ethanol are oriented for direct hydrogen transfer when they are bound to the enzyme LADH. In this enzyme a zinc atom in the catalytic domain binds the alcohol group of the substrate. This zinc is essential for catalysis in LADH both by binding the alcohol and by participating in the abstraction of the proton and, in addition, by polarizing the C-O bond that facilitates hydrogen transfer. The catalytic mechanisms are different in LDH and GAPDH, neither of which contains zinc. The NAD-binding domains have similar structures Despite the absence of amino acid sequence homology, the NAD-binding domains of LDH, GAPDH, and LADH have very similar three-dimensional Figure 10.5 (a) The subunits of NAD-dependent dehydrogenases are folded into two separate domains, one of which, the dinucleotide-binding domain, binds the cofactor NAD. The second domain is responsible for the specificity of substrate binding and also provides the catalytic groups. The diagrams (b) and (c) illustrate schematically the domain organization o the subunit structures of liver alcohol dehydrogenase (b) and glyceraldehyde-phosphate dehydrogenase (c). The two grey balls in (b) represent zinc atoms. The catalytic zinc atom is close to the cofactor, NAD. [(b) Adapted from H. Eklund et al., /. Mol. Biol. 146: 561, 1981 and diagram (c) from G. Biesecker et al., Nature 266: 328,
structures. The catalytic domains, on the other hand, have completely different structures in these three enzymes; indeed, each has a unique topology, which has not yet been observed in other proteins. The NAD-binding domain is an open, parallel six-stranded ß sheet with helices on both sides of the sheet (Figure 10.7a and c). This α/β structure is symmetrical because it is built from two halves with identical topology and similar structure (Figure 10.7b). Each half is formed by a pair of β-α-β motifs, as shown in Figure 10.7b. A crossover connection (yellow in Figure 10.7a) links the two halves, which are joined by hydrogen bonds between β strands 1 and 4 into a sixstranded β sheet. Each half of this symmetrical domain is called a mononucleotide-binding motif for two reasons. First, each half of the NADbinding domain binds one of the two nucleotides in the dinucleotide NAD. Second, the half structure occurs in proteins that bind mononucleotides, for example, flavodoxin, which binds FMN. Not only do the NAD-binding domains of LDH, GAPDH, and LADH have identical topology, but large parts of their actual structures are so similar that many of their main chain atoms superimpose within 2 Å. These superimposable parts are colored in figure 10.8; they comprise the complete β1-αa-β2 motif including the loop regions, the major parts of a helices B and D, and the remaining four β strands. The other regions of the domain are of different lengths and conformations, but the total lengths of the NAD-binding domains are about the same in all these enzymes, around 140 amino acids. Figure 10.6 The NAD-binding domains (red boxes) of the three dehydrogenases GAPDH, LDH, and LADH are located at different positions within the polypeptide chains of these enzymes. In GAPDH and LDH they are in the N termini of the chains, whereas in LADH this domain is within the C-terminal region. Figure 10.7 The NAD-binding domains have similar three-dimensional structures in spite of their completely different amino acid sequences. The structure is of the α/β type with an open twisted parallel β sheet in the middle surrounded by helices on both sides and is divided into two similar halves (red and green). These are called mononucleotide-binding motifs or Rossmann folds, after Michael Rossmann, who first pointed out that this was a frequently occurring motif in nucleotidebinding proteins. The diagrams show an idealized NAD-binding motif (a) and its topology diagrams (b and c).
Figure 10.8 The six β strands and three of the four a helices form a common structural framework (red) in the NAD-binding domains of LADH, LDH, and GAPDH. The diagrams illustrate this framework viewed along the β sheet in the actual structures of LADH (a) and LDH (b). The regions outside this common framework have quite different structures in these two enzymes. (Adapted from J. Richardson.) NAD binds in a similar way to each domain In all three enzymes the regions of similar structure in the NAD-binding domains form the structural framework of the main chain that binds the NAD molecule. The actual interactions between the NAD and the protein mainly occur, however, through amino acid side chains. Two things are remarkable about this binding. First, the NAD molecules bind in almost identical positions in the three proteins, and the conformation of the NAD molecule itself is the same in all three cases. This conformation is not the most stable conformation for free NAD, as shown by theoretical calculations; nor is it the conformation found in crystals of NAD. It, therefore, follows that interaction with the protein forces NAD into an energetically unfavorable conformation. Second, NAD binding involves different combinations of side chains in different enzymes. In fact, comparisons of even the same enzyme, LADH, from different species have shown that these side chains vary between species almost as frequently as nonfunctional amino acids at the surface of the protein (Figure 10.9). In other words, there are several equally good ways of reaching a common structural goal, and this is reflected in the evolution of these enzymes. The NAD-binding cleft is located outside the carboxy ends of β strands 1 and 4 (Figure 10.10). This is where the strand order is reversed. Thus, the binding cleft is precisely where it would be predicted in this α/β structure following the rule discussed in Chapter 4. As Figure 10.10 shows, the pyrophosphate group in Figure 10.9 Amino acid sequences of alcohol dehydrogenases from different species. Residuthat participate in NAD binding (orange) are not invariant but vary within these species almost as frequently as nonfunctional residue-
Figure 10.10 The coenzyme NAD is bound to the NAD-binding domain outside the carboxy edge of the parallel β sheet with the pyrophosphate group straddling the sheet and the two ends on opposite sides of the β sheet. The diagram shows the coenzyme viewed from the top of the four middle β strands of the domain. The loop regions from these strands form a crevice where the pyrophosphate group is bound. the middle of NAD binds to the central region of the domain straddling the β sheet. The nucleosides (see Figure 10.1a) on the flanks of NAD bind outside the β strands to opposite sides of the β sheet. Adenosine binds to the first mononucleotide-binding motif and nicotinamide ribose to the second motif. Hydride transfer to NAD is stereospeciflc There are many enzymatic reactions in which the enzyme distinguishes between the stereospeciflc isomers of its substrate and binds only one of them. The dehydrogenases, however, are the classical example of another type of stereospecificity where the enzyme transfers a group (a hydrogen atom) in a stereospeciflc manner between two molecules. We now understand the structural basis of this stereospecificity. For many years the NAD-dependent dehydrogenases have been divided into two classes, A and B, depending on the stereospecificity of hydrogen transfer to the C4 atom of the nicotinamide ring (Figure 10.11). The ring is asymmetric because of the carboxamide substituent at its C3 atom. The two hydrogen atoms at C4 in the reduced form of NAD (above and below the ring in Figure 10.11) are therefore not equivalent. The two positions can be distinguished by using a deuterated substrate so that the enzyme transfers a deuterium atom to NAD instead of a hydrogen atom. LADH and LDH belong to class A because the hydrogen atom is transferred from the substrate to the position above the plane of the ring in Figure 10.11 whereas GAPDH belongs to class B and transfers hydrogen to the position below the ring. Many theories have been proposed, and many experiments performed, to try to deduce a fundamental mechanistic or metabolic reason for the existence of these two classes of dehydrogenases. The structures of these enzymes provide a simple and trivial answer; this difference merely reflects the different structures of the catalytic domains. The stereochemistry is preserved for a specific enzyme within different species because the conformation of the enzyme is essentially preserved. Figure 10.11 (a) Stereospecificity of hydrogen transfer to the nicotinamide part of the coenzyme NAD. In the A-form the transferred hydrogen is above the plane of the ring (red) and in the B-form it is below (green) when the carboxamide group is oriented, as shown in this diagram, (b) Structural basis for the stereospecificity of hydrogen transfer in dehydrogenases. Each enzyme has a pocket for binding the carboxamide group so that only one face of the nicotinamide ring is available for hydride transfer.
In all three enzymes the nicotinamide ring of the coenzyme is positioned in a cleft between the two domains close to the substrate that is bound to the catalytic domain. One side of the ring interacts with the structural framework of the NAD-binding domain, and the other side faces the substrate binding site (Figure 10.11). Interactions between the carboxamide group of NAD and the region of the protein that links the two domains determines if the A or the B side of the nicotinamide ring faces the substrate and therefore determines the stereospecificity of the transfer of the hydrogen atom. The conformational difference between the two states of NAD is a simple flip of the nicotinamide ring 180 around the glycosidic bond that links it to the ribose. This difference positions the carboxamide group in two different ways. Therefore, depending on the relative orientation in the enzyme of the two domains and the side chains in the region that joins them, the carboxamide group makes a better fit with the A side of the nicotinamide ring facing the substrate in some enzymes and with the B side in others. In this way the structure of the enzyme discriminates between the two stereochemical forms of NAD. Are the NAD-binding domains evolutionary related? In Chapter 4 we discussed the evolution of α/β-barrel structures and reached the conclusion that they probably were not derived from a common ancestral gene. The barrel structures are similar, but their functions are not. The situation is different for the NAD-binding domains we have just discussed because both structure and function are preserved. The structural framework is similar as well as the conformation of bound NAD, which positions the nicotinamide ring close to the substrate. Yet, most of the side chains that interact with NAD can vary. This is clear from comparisons of the amino acid sequences of LADH from different species (Figure 10.9). Many different combinations of side chains can form this framework and preserve the function. The functionally important aspect of these domains is, therefore, the structural framework and not individual amino acid residues, except those that are necessary to preserve the structure. The essential lesson of these enzymes is that similar structural frameworks can be obtained from many different amino acid sequences. It is, therefore, impossible to decide with any degree of confidence between the two possible evolutionary histories of the NAD-binding domains, namely, convergent evolution from different ancestral genes or divergent evolution from a common ancestor. It is, however, attractive to speculate that the dehydrogenases arose by the fusion of a gene for an ancestral NAD-binding protein with genes for primitive proteins able to bind different metabolites, such as alcohol, lactate, malate, glyceraldehyde-3-phosphate, and so on. The fusion of genes coding small proteins with single functions to generate a greater variety of more complex multifunctional proteins is a well-demonstrated evolutionary mechanism (recall DNA polymerase I) and occurs all the time in the somatic cells of the immune system, as will be discussed in Chapter 12. The NAD-binding motif can be predicted from amino acid sequence The absence of significant amino acid sequence homologies between even the NAD-binding domains of the different dehydrogenases means that we cannot say anything about the evolutionary histories of these proteins. We have remarked that the apparent nonconservation of the amino acid sequences of the proteins extends to the site of NAD binding. But although most of the residues at this site can vary, there are certain key invariant residues that make it possible to predict from the amino acid sequence the regions of the polypeptide chains that are involved in NAD binding. The reason for this is that in the NAD-binding domain, as in the calcium-binding and DNA-binding motifs discussed earlier, there are strong stereochemical constraints at specific positions in the polypeptide chain that must be respected to preserve the structure and function of the domain. The amino acids at these key sites are diagnostic.
1 Horse liver alcohol dehydrogenase (194-224) 2 Dogfish muscle lactate dehydrogenase (22-53) 3 Lobster glyceraldehyde-3-phosphate dehydrogenase (2-32) 4 Human erythrocyte glutathione reductase (22-51) 5 Pseudomonas fluorescens p-hydroxybenzoate hydroxylase (4-32) 6 Drosophila alcohol dehydrogenase (9-38) 7 Bovine glutamate dehydrogenase (246-275) 8 Rabbit muscle glycerol-3-phosphate dehydrogenase (4-33) 9 Agrobacterium tumefaciens nopaline synthase (34-45) 10 Pig L'3-hydroxyacyl-CoA-dehydrogenase (17-45) 11 Pig D-amine oxidase (2-31) Figure 10.12 (a) Schematic diagram of the β1- αa-β2 motif in dinucleotide-binding proteins that has been used to derive a fingerprint to predict dinucleotide-binding regions in proteins of known amino acid sequence. The sequence shown here is from LDH. Hydrophobie side chains are required at certain positions (green) for packing the β strands against the a helix. Three invariant glycine residues (yellow) are required to form a tight loop and to bring the ADP part of the coenzyme NAD (thin lines) in close contact with the main chain of this loop. The motif comprises 31 amino acid residues with its amino end at position 22 and the carboxy end at Asp 52 (red), which forms a hydrogen bond to the 2'-OH of the adenosine ribose. (b) Amino acid sequences of β1-αa-β2 motifs. Sequences 1-5 are from known structures that were used to derive the fingerprint for either NAD or FAD binding. Proteins 1-3 bind NAD, 4 and 5 bind FAD. The remaining dinucleotide-binding regions were predicted using this fingerprint. (Adapted from R. Wierenga et al., /. Mol. Biol. 187: 101, 1986.)
One region in particular, the β1-αa-β2 motif, has a highly conserved structure and has been used to identify NAD-binding regions in enzymes of unknown three-dimensional structure (Figure 10.12a). In this motif, comprising about 30 amino acids, there are three conserved glycine residues (yellow in the figure) with the sequence Gly-X-Gly-X-X-Gly, where X is any residue, and there are six conserved hydrophobic residues (green in the figure), which form a hydrophobic core between the helix and the β strands, and finally, there is one conserved Asp (red in the figure) at the carboxy end of β2. The glycine-rich region plays a crucial role in positioning the central part of NAD in its correct conformation close to the protein framework. This region forms the loop between β1 and α-helix A as well as the first residues of αa (Figure 10.12a).The loop region is short and very close to the strand and the helix. The amino end of α-helix A makes hydrogen bonds to the pyrophosphate group and thereby positions it as described in Chapter 2 (Figure 2.3c). Large side chains at these glycine positions would either disturb the structure of this framework or, in the case of glycines 27 and 29, push NAD out from the framework into an incorrect position. The conserved aspartate has a special function. It is the principal means by which these enzymes discriminate between NAD and NADP as coenzyme. NAD binds to LDH, GAPDH, and LADH through hydrogen bonds between the 2'-OH of its adenosine ribose and the conserved Asp in the enzyme. But NADP has a phosphate group attached to the 2'-OH of the adenosine ribose (Figure 10.1b), and its binding to these enzymes is thus prevented by the repulsion between the negative charges of the phosphate group in the 2' position and the conserved Asp. We would expect that enzymes that bind NADP instead of NAD by this motif should have a small side chain instead of the Asp residue to make room for the phosphate group and a positively charged side chain nearby for binding the phosphate. Threonine dehydrogenase from E. coli, which is homologous to LADH (25% identity) and therefore has a similar structure including the β-α-β motif, uses NADP as coenzyme. As expected, this enzyme has a glycine residue instead of Asp and an Arg instead of Asn in a nearby position where the positive charge can interact with the phosphate group. These stereochemical constraints, which are specific for enzymes that bind dinucleotides of which one-half is AMP, have been used to predict the NADbinding regions in several dehydrogenases. Some of these sequences are given Figure 10.13 (a) The FAD requiring enzymes glutathione reductase and p-hydroxybenzoate hydroxylase are dimeric molecules where each subunit is divided into three domains FADbinding domain (FAD), NADPH-binding domain (NADPH), and subunit interface domain (Int). The diagram shows the domain arrangement in glutathione reductase. The red circles indicate the positions of the active sites. (Adapted from E.F. Pai and G. Schulz, J. Biol. Chem. 258: 1752, 1983.) (b) Glutathione reductase is a flavoenzyme that catalyzes reduction of the S-S bridge that links the two halves of the molecule in oxidized glutathione (GSSG). Each half derives from a tripeptide, NH 2 -Glu-Cys-Gly-COOH, which is the reduced form of glutathione (GSH). The carboxy side chain of Glu forms the peptide link to the amino group of Cys in the tripeptide. During reduction the electrons flow from NADPH to oxidized glutathione through FAD and a redoxactive S-S bridge which is formed by two Cys residues of the enzyme, (c) p-hydroxybenzoate hydroxylase is a flavoenzyme that catalyzes the addition of a hydroxyl group to p- hydroxybenzoate in a reaction that requires oxygen. During hydroxylation the electrons flow from NADPH through FAD and oxygen to the substrate, p-hydrozybenzoate. Although the reactions catalyzed by this enzyme and glutathione reductase are quite different, their FAD-binding domains have similar threedimensional structures.
in Figure 10.12b. The structures of these proteins are not yet known, but analysis of mutants has confirmed the prediction in at least one case, Drosophila alcohol dehydrogenase. This enzyme has a completely different amino acid sequence and a different catalytic mechanism from that of LADH even though the two enzymes catalyze the same reaction. In one mutant the first invariant glycine was changed to Asp. The mutant enzyme is inactive, and this poor fly would not be able to thrive in Spanish wine cellars like its wild-type relatives.