25.1 Which Metabolic Pathways Allow Organisms to Live on Inorganic Forms of Nitrogen? 773 tase, so it no longer binds to nitrogenase. The ADPϺoxidized nitrogenase reductase complex dissociates, making way for another ATPϺreduced nitrogenase reductase complex to bind to nitrogenase. Interestingly, nitrogenase reductase is a member of the G-protein family; G proteins are molecular switches whose operation is driven by NTP hydrolysis. Nitrogenase is a rather slow enzyme: Its optimal rate of e Ϫ transfer is about 12 e Ϫ pairs per second per enzyme molecule; that is, it reduces only three molecules of nitrogen gas per second. Because its activity is so weak, nitrogen-fixing cells main- tain large amounts of nitrogenase so that their requirements for reduced N can be met. As much as 5% of the cellular protein may be nitrogenase. The Regulation of Nitrogen Fixation To a first approximation, two regulatory controls are paramount (Figure 25.8): (1) ADP inhibits the activity of nitrogenase; thus, as the ATP/ADP ratio drops, nitrogen fixation is blocked. (2) NH 4 ϩ represses FIGURE 25.7 Ribbon diagram of nitrogenase reductase (the Fe-protein, blue)Ϻnitrogenase (FeMo protein, green) complex.The Fe-protein iron-sulfur cluster is shown in yellow, bound ADP in orange.The nitrogenase FeMo cofactor is shown in cyan, the P-cluster in red (pdb id ϭ 1N2C). Nitrogenase Nitrogenase reductase N NH 2 C O Nitrogenase reductase Arg 101 O ADP e – nif gene expression N 2 + + 10 H + 2 NH 4 + + H 2 16 16 P i + 16 ADP (c) (b) (a) Nicotinamide Ribose Arg 101 H 2 O Active Inactive ADP–ribosyl g roup ATP NAD + ADP 8 FIGURE 25.8 Regulation of nitrogen fixation. (a) ADP inhibits nitrogenase activity. (b) NH 4 ϩ represses nif gene expression. (c) In some organisms, the nitrogenase complex is regulated by covalent modification. ADP– ribosylation of nitrogenase reductase leads to its inactivation. 774 Chapter 25 Nitrogen Acquisition and Amino Acid Metabolism the expression of the nif genes, the genes that encode the proteins of the nitrogen- fixing system. To date, some 20 nif genes have been identified with the nitrogen fix- ation process. Repression of nif gene expression by ammonium, the primary prod- uct of nitrogen fixation, is an efficient and effective way of shutting down N 2 fixation when its end product is not needed. In addition, in some systems, covalent modifi- cation of nitrogenase reductase leads to its inactivation. Inactivation occurs when Arg 101 of nitrogenase reductase receives an ADP-ribosyl group donated by NAD ϩ . 25.2 What Is the Metabolic Fate of Ammonium? Given the prevalence of N atoms in cellular components, it is surprising that only three enzymatic reactions introduce ammonium into organic molecules. Of these three, glutamate dehydrogenase and glutamine synthetase are responsible for most of the ammonium assimilated into carbon compounds. The third, carbamoyl-phosphate syn- thetase I, is a mitochondrial enzyme that participates in the urea cycle. Glutamate dehydrogenase (GDH) catalyzes the reductive amination of ␣-keto- glutarate to yield glutamate. Reduced pyridine nucleotides (NADH or NADPH) pro- vide the reducing power: NH 4 ϩ ϩ ␣-ketoglutarate ϩ NADPH ϩ H ϩ ⎯⎯→ glutamate ϩ NADP ϩ ϩ H 2 O (25.6) This reaction provides an important interface between nitrogen metabolism and cel- lular pathways of carbon and energy metabolism because ␣-ketoglutarate is a citric acid cycle intermediate. In vertebrates, GDH is an ␣ 6 -type multimeric enzyme localized in the mitochondrial matrix that uses NADPH as electron donor when operating in the biosynthetic direction (the direction of glutamate synthesis) (Figure 25.9). In contrast, when GDH acts in the catabolic direction to generate ␣-ketoglutarate from glutamate, NAD ϩ , not NADP ϩ , is usually the electron acceptor. The catabolic activity is allosteri- cally activated by ADP and inhibited by GTP. Glutamine synthetase (GS) catalyzes the ATP-dependent amidation of the ␥-carboxyl group of glutamate to form glutamine (Figure 25.10). The reaction pro- ceeds via a ␥-glutamyl-phosphate intermediate, and GS activity depends on the presence of divalent cations such as Mg 2ϩ . Glutamine is a major N donor in the biosynthesis of many organic N compounds such as purines, pyrimidines, and other amino acids, and GS activity is tightly regulated, as we shall soon see. The amide-N of glutamine provides the nitrogen atom in these biosyntheses. Carbamoyl-phosphate synthetase I, the third enzyme capable of using ammo- nium to form an N-containing organic compound, catalyzes an early step in the urea cycle. Two ATPs are consumed, one in the activation of HCO 3 Ϫ for reaction with ammonium and the other in the phosphorylation of the carbamate formed (see also Figure 25.22): (25.7) N-acetylglutamate is an essential allosteric activator for this enzyme. NH 4 ϩ ϩ HCO 3 Ϫ ϩ 2 ATP H 2 N C O PO 3 2Ϫ ϩ 2 ADP ϩ P i ϩ 2 H ϩ O NH 3 + O O – C CH 2 CH 2 C O C OO – + O O – C CH 2 CH 2 C OO – HC NH 4 + Glu H 2 O ␣-KG NADPH NADP + FIGURE 25.9 The glutamate dehydrogenase reaction. 25.2 What Is the Metabolic Fate of Ammonium? 775 The Major Pathways of Ammonium Assimilation Lead to Glutamine Synthesis In organisms that enjoy environments rich in nitrogen, GDH and GS acting in se- quence furnish the principal route of NH 4 ϩ incorporation (Figure 25.11). However, GDH has a significantly higher K m for NH 4 ϩ than does GS. Consequently, in organ- isms such as green plants that grow under conditions where little NH 4 ϩ is available, GDH is not effective and GS is the only NH 4 ϩ -assimilative reaction. Such a situation creates the need for an alternative mode of glutamate synthesis to replenish the glu- tamate consumed by the GS reaction. This need is filled by glutamate synthase (also known as GOGAT, the acronym for the other name of this enzyme—glutamateϺ oxo-glutarate amino-transferase). Glutamate synthase catalyzes the reductive amina- tion of ␣-ketoglutarate using the amide-N of glutamine as the N donor: Reductant ϩ ␣-KG ϩ Gln ⎯⎯→ 2 Glu ϩ oxidized reductant (25.8) Two glutamates are formed—one from amination of ␣-ketoglutarate and the other from deamidation of Gln (Figure 25.12). These glutamates can now serve as am- monium acceptors for glutamine synthesis by GS. Organisms variously use NADH, NADPH, or reduced ferredoxin as reductant. Glutamate synthases are typically large, complex proteins; in Escherichia coli, GOGAT is an 800-kD flavoprotein con- taining both FMN and FAD, as well as [4Fe-4S] clusters. NH 3 + O O – C CH 2 CH 2 C C OO – ++ + O C CH 2 CH 2 C C OO – + H NH 4 + NH 3 + H NH 2 O O – C CH 2 CH 2 C C OO – + NH 3 + H NH 3 + O CH 2 CH 2 C C C OO – H O – O P O – O NH 4 + O – O P OH HO NH 3 + O C CH 2 CH 2 C C OO – H NH 2 ATP ATP ADP P i Gln (a) Glu Mg 2+ Glu ␥-Glutamyl-P (b) Gln ADP (a) (b) FIGURE 25.10 (a) The enzymatic reaction catalyzed by glutamine synthetase. (b) The reaction proceeds by (a) activation of the ␥-carboxyl group of Glu by ATP, fol- lowed by (b) amidation by NH 4 ϩ . (a) NH 4 + + ␣-ketoglutarate + NADPH glutamate + NADP + + H 2 O GDH (b) Glutamate + NH 4 + + ATP glutamine + ADP + P i GS SUM: 2 NH 4 + + ␣-ketoglutarate + NADPH + ATP glutamine + NADP + + ADP + P i + H 2 O FIGURE 25.11 The GDH/GS pathway of ammo- nium assimilation.The sum of these reactions is the conversion of 1 ␣-ketoglutarate to 1 glu- tamine at the expense of 2 NH 4 ϩ , 1 ATP, and 1 NADPH. 776 Chapter 25 Nitrogen Acquisition and Amino Acid Metabolism Together, GS and GOGAT constitute a second pathway of ammonium assimilation, in which GS is the only NH 4 ϩ -fixing step; the role of GOGAT is to regenerate gluta- mate (Figure 25.13). Note that this pathway consumes 2 equivalents of ATP and 1 NADPH (or similar reductant) per pair of N atoms introduced into Gln, in contrast to the GDH/GS pathway, in which only 1 ATP and 1 NADPH are consumed per pair of NH 4 ϩ fixed. Clearly, coping with a nitrogen-limited environment has its cost. 25.3 What Regulatory Mechanisms Act on Escherichia coli Glutamine Synthetase? As indicated earlier, glutamine plays a pivotal role in nitrogen metabolism by donating its amide nitrogen to the biosynthesis of many important organic N com- pounds. Consistent with its metabolic importance, in prokaryotic cells such as E. coli, GS is regulated at three different levels: 1. Its activity is regulated allosterically by feedback inhibition. 2. GS is interconverted between active and inactive forms by covalent modification. 3. Cellular amounts of GS are carefully controlled at the level of gene expression and protein synthesis. Eukaryotic versions of glutamine synthetase show none of these regulatory features. E. coli GS is a 600-kD dodecamer (␣ 12 -type subunit organization) of identical 52-kD monomers (each monomer contains 468 amino acid residues). These monomers are arranged as a stack of two hexagons (Figure 25.14). The active sites are located at subunit interfaces within the hexagons; these active sites are recognizable in the X-ray crystallographic structure by the pair of divalent ␣-KG NH 3 + O O – C CH 2 CH 2 C O C OO – + O O – C CH 2 CH 2 C C OO – H Gln NH 3 + O C CH 2 CH 2 C C OO – H NH 2 NADH (yeast, N. crassa) + H + NADPH (E. coli) + H + or 2 H + + 2 reduced ferredoxin (plants) + ␣-KG + Gln 2 Glu + NAD + NADP + or 2 oxidized ferredoxin NH 3 + O O – C CH 2 CH 2 C C OO – H + Glu Glu (a) 2 NH 4 + + 2 ATP + 2 glutamate 2 glutamine + 2 ADP + 2 P i GS (b) NADPH + ␣-ketoglutarate + glutamine 2 glutamate + NADP + GOGAT SUM: 2 NH 4 + + ␣-ketoglutarate + NADPH + 2 ATP glutamine + NADP + + 2 ADP + 2P i FIGURE 25.12 The glutamate synthase reaction (left), showing the reductants exploited by different organisms in this reductive amination reaction. Structure of glutamate synthase (right) (pdb id ϭ 1LM1) FAD is shown in blue, the Fe-S cluster in yellow. FIGURE 25.13 The GS/GOGAT pathway of ammonium assimilation.The sum of these reactions results in the conversion of 1 ␣-ketoglutarate to 1 glutamine at the expense of 2 ATP and 1 NADPH. 25.3 What Regulatory Mechanisms Act on Escherichia coli Glutamine Synthetase? 777 cations that occupy them. Adjacent subunits contribute to each active site, thus accounting for the fact that GS monomers are catalytically inactive. Glutamine Synthetase Is Allosterically Regulated Nine distinct feedback inhibitors (Gly, Ala, Ser, His, Trp, CTP, AMP, carbamoyl-P, and glucosamine-6-P) act on GS. Gly, Ala, and Ser are key indicators of amino acid me- tabolism in the cell; each of the other six compounds represents an end product of a biosynthetic pathway dependent on Gln (Figure 25.15). AMP competes with ATP for binding at the ATP substrate site. Gly, Ala, and Ser compete with Glu for binding at the active site. Carbamoyl-P binds at a site that overlaps both the Glu site and the site occupied by the ␥-PO 4 of ATP. Glutamine Synthetase Is Regulated by Covalent Modification Each GS subunit can be adenylylated at a specific tyrosine residue (Tyr 397 ) in an ATP-dependent reaction (Figure 25.16). Adenylylation inactivates GS. If we de- fine n as the average number of adenylyl g roups per GS molecule, GS activity is inversely proportional to n. The number n varies from 0 (no adenylyl groups) to 12 (every subunit in each GS molecule is adenylylated). Adenylylation of GS is catalyzed by the converter enzyme ATPϺGSϺadenylyl transferase, or simply adenylyl transferase (AT). However, whether or not this covalent modification occurs is de- termined by a highly regulated cycle (Figure 26.17). AT not only catalyzes adeny- lylation of GS, it also catalyzes deadenylylation—the phosphorolytic removal of the Tyr-linked adenylyl groups as ADP. The direction in which AT operates de- pends on the nature of a regulatory protein, P II , associated with it. P II is a 44-kD protein (tetramer of 11-kD subunits): The state of P II controls the direction in (a) (b) FIGURE 25.14 The subunit organization of bacterial glutamine synthetase. (a) Schematic; (b) molecular structure (note the pairs of metal ions [dark blue] that define the active sites) (pdb id ϭ 1FPY). 778 Chapter 25 Nitrogen Acquisition and Amino Acid Metabolism P R O O – + H 3 N Mg 2+ C H H C O O – + H 3 NC CH 3 H C Glutamine O O – + H 3 NC H C CH 2 OH O O – CH CCH 2 NNH N H O O – C H CCH 2 + NH 3 O NH 2 CH 2 P P P R N N NH 2 N N N N NH 2 O – O P O – O O C + H 3 N P + NH 3 O ATP P Glutamate +NH 4 + + Glycine Alanine Glutamine synthetase Serine Histidine Tryptophan Glucosamine-6-P CTP Carbamoyl-P ADP AMP FIGURE 25.15 The allosteric regulation of glutamine synthetase activity by feedback inhibition. CH 2 P P OH CH 2 O – OP O OCH 2 O N HO OH N N N NH 2 ATP 12 + Tyr 397 in GS monomer Glutamine synthetase monomer Adenylyl transferase 12 + Glutamine synthetase monomer 12 12 Adenylylated Tyr 397 in GS monomer FIGURE 25.16 Covalent modification of GS: Adenylylation of Tyr 397 in the glutamine synthetase polypeptide via an ATP-dependent reaction catalyzed by the converter enzyme adenylyl transferase. 25.4 How Do Organisms Synthesize Amino Acids? 779 which AT acts. If P II is in its so-called P IIA form, the ATϺP IIA complex acts to adenylylate GS. When P II is in its so-called P IID form, the ATϺP IID complex cat- alyzes the deadenylylation of GS. The active sites of ATϺP IIA and ATϺP IID are dif- ferent, consistent with the difference in their catalytic roles. In addition, the ATϺP IIA and ATϺP IID complexes are allosterically regulated in a reciprocal fash- ion by the effectors ␣-KG and Gln. Gln activates ATϺP IIA activity and inhibits ATϺP IID activity; the effect of ␣-KG on the activities of these two complexes is di- ametrically opposite (Figure 25.17). Further, Gln favors conversion of P IID to P IIA , whereas ␣-ketoglutarate favors the P IID over the P IIA form. Clearly, the determining factor regarding the degree of adenylylation, n, and hence the relative activity of GS, is the [Gln]/[␣-KG] ratio. A high [Gln] level sig- nals cellular nitrogen sufficiency, and GS becomes adenylylated and inactivated. In contrast, a high [␣-KG] level is an indication of nitrogen limitation and a need for ammonium fixation by GS. Glutamine Synthetase Is Regulated Through Gene Expression The gene that encodes the GS subunit in E. coli is designated GlnA. The GlnA gene is actively transcribed to yield GS mRNA for translation and synthesis of GS protein only if a specific transcriptional enhancer, NR I , is in its phosphorylated form, NR I -P. In turn, NR I is phosphorylated in an ATP-dependent reaction catalyzed by NR II , a pro- tein kinase (Figure 25.18). However, if NR II is complexed with P IIA , it acts not as a kinase but as a phosphatase, and the transcriptionally active form of NR I , namely NR I -P, is converted back to NR I with the result that GlnA transcription halts. Recall from the foregoing discussion that a high [Gln]/[␣-KG] ratio favors P IIA at the ex- pense of P IID . Under such conditions, GS gene expression is not necessary. 25.4 How Do Organisms Synthesize Amino Acids? Organisms show substantial differences in their capacity to synthesize the 20 amino acids common to proteins. Typically, plants and microorganisms can form all of their nitrogenous metabolites, including all of the amino acids, from P P P + + ATP 12 12 12 Adenylyl transferase: P IIA complex Adenylyl transferase: P IID complex 12 ADP Less active GS: Tyr 397 –O– AMP adenylylated ␣-KG Gln ␣-KG Gln Active GS: Tyr 397 unadenylylated Highly sensitive to feedback inhibition *State of P II controls adenylyl transferase direction FIGURE 25.17 The cyclic cascade system regulating the covalent modification of GS. + NR II H 2 O P NR I NR I — NR II :P IIA P activates GlnA transcription; GS is synthesized ATP ADP FIGURE 25.18 Transcriptional regulation of GlnA expres- sion through the reversible phosphorylation of NR I . 780 Chapter 25 Nitrogen Acquisition and Amino Acid Metabolism inorganic forms of N such as NH 4 ϩ and NO 3 Ϫ . In these organisms, the ␣-amino group for all amino acids is derived from glutamate, usually via transamination of the corresponding ␣-keto acid analog of the amino acid (Figure 25.19). In many cases, amino acid biosynthesis is thus a matter of synthesizing the appropriate ␣-keto acid carbon skeleton, followed by transamination with Glu. The amino acids can be classified according to the source of intermediates for the ␣-keto acid biosynthesis (Table 25.1). For example, the amino acids Glu, Gln, Pro, and Arg CH 2 C COO – NH 3 + COO – COO – COO – COO – COO – COO – COO – COO – COO – COO – CH 2 CH 2 CH + R O CH 2 CH 2 C O C COO – + R H NH 3 + NH 3 + CH 2 CH 2 CH + C COO – O CH 2 CH 2 C O + C COO – H NH 3 + CH 2 Glutamate ␣-Keto acid ␣-KG ␣-Amino acid Glutamate ␣-KG Oxaloacetate Aspartate Pyridoxal phosphate- dependent aminotransferase Glutamate- aspartate aminotransferase ACTIVE FIGURE 25.19 Glutamate- dependent transamination of ␣-keto acid carbon skele- tons is a primary mechanism for amino acid synthesis. The transamination of oxaloacetate by glutamate to yield aspartate and ␣-ketoglutarate is a prime example. Test yourself on the concepts in this figure at www.cengage.com/login. ␣-Ketoglutarate Family Aspartate Family Glutamate Aspartate Glutamine Asparagine Proline Methionine Arginine Threonine Lysine* Isoleucine Lysine* Pyruvate Family 3-Phosphoglycerate Family Alanine Serine Valine Glycine Leucine Cysteine Phosphoenolpyruvate and Erythrose-4-P Family The aromatic amino acids Phenylalanine Tyrosine Tryptophan The remaining amino acid, histidine, is derived from PRPP (5-phosphoribosyl-1-pyrophosphate) and ATP. *Different organisms use different precursors to synthesize lysine. TABLE 25.1 The Grouping of Amino Acids into Families According to the Metabolic Intermediates That Serve as Their Progenitors 25.4 How Do Organisms Synthesize Amino Acids? 781 (and, in some instances, Lys) are all members of the ␣-ketoglutarate family be- cause they are all derived from the citric acid cycle intermediate ␣-ketoglutarate. We return to this classification scheme later when we discuss the individual biosynthetic pathways. Amino Acids Are Formed from ␣-Keto Acids by Transamination Transamination involves transfer of an ␣-amino group from an amino acid to the ␣-keto position of an ␣-keto acid (Figure 25.19). In the process, the amino donor be- comes an ␣-keto acid while the ␣-keto acid acceptor becomes an ␣-amino acid: Amino acid 1 ϩ ␣-keto acid 2 ⎯⎯→␣-keto acid 1 ϩ amino acid 2 (25.9) HUMAN BIOCHEMISTRY Human Dietary Requirements for Amino Acids Humans can synthesize only 10 of the 20 common amino acids (see table below); the others must be obtained in the diet. Those that can be synthesized are classified as nonessential, meaning it is not essential that these amino acids be part of the diet. In ef- fect, humans can synthesize the ␣-keto acid analogs of nonessen- tial amino acids and form the amino acids by transamination. In contrast, humans are incapable of constructing the carbon skele- tons of essential amino acids, so they must rely on dietary sources for these essential metabolites. Excess dietary amino acids cannot be stored for future use, nor are they excreted unused. Instead, they are converted to common metabolic intermediates that can be either oxidized by the citric acid cycle to generate metabolic energy or used to form glucose (see Section 25.5). Since autotrophic cells (and many prokaryotic cells) synthesize all 20 amino acids, several questions arise regarding human di- etary requirements for amino acids. First, why is it that humans lack the ability to do what other organisms can do? The answer is that, over evolutionary time, human diets provided adequate amounts of those amino acids classified now as “essential.” Thus, the loss of the metabolic pathways for synthesis of “essential” amino acids did not impair the fitness of humans. That is, synthe- sizing “essential” amino acids became superfluous, and no evolu- tionary pressure operated on humans to retain the genes for these pathways. A second question now emerges: Are there significant differences between synthesis of amino acids human can make (the so-called “nonessential” amino acids) and those they can’t? The big table summarizes amino acid biosynthesis from citric acid cycle intermediates in terms of the number of reactions needed to make an amino acid from a TCA cycle intermediate.* Nonessen- tial amino acids are shown in blue; essential amino acids in red. Two conclusions stand out: Nonessential amino acids require fewer reaction steps for synthesis than essential amino acids, and nonessential amino acids tend to be more abundantly represented in proteins than essential amino acids. Thus, evolutionary loss was not random: The biosynthetic pathways lost were those for amino acids requiring the most reaction steps. *From Srinivasan, V., Morowitz, H., and Smith, E., 2007. Essential amino acids, from LUCA to LUCY. Complexity 13:8–9. Essential Nonessential Arginine* Alanine Histidine* Asparagine Isoleucine Aspartate Leucine Cysteine Lysine Glutamate Methionine Glutamine Phenylalanine Glycine Threonine Proline Tryptophan Serine Valine Tyrosine† *Arginine and histidine are essential in the diets of juveniles, not adults. †Tyrosine is classified as nonessential only because it is readily formed from essential phenylalanine. Essential and Nonessential Amino Acids in Humans Amino Acid Reaction Steps Mole % in Proteins† 1 Alanine 1 7.9 2 Aspartic acid 1 5.3 3 Glutamic acid 1 6.7 4 Asparagine 2 4.1 5 Glutamine 2 4.0 6 Serine 5 6.9 7 Glycine 6 6.9 8 Proline 6 4.8 9 Cysteine 7 1.5 10 Threonine 6 5.4 11 Valine 9 6.7 12 Isoleucine 13 5.9 13 Leucine 14 10.0 14 Lysine 14 5.9 15 Methionine 17 2.4 16 Arginine 24 5.4 17 Histidine 27 2.3 18 Phenylalanine 29 4.0 19 Tyrosine‡ 30 3.0 20 Tryptophan 33 1.1 †Mole percentages are taken from amino acid representations among proteins in the Swiss-Prot protein knowledgebase: ca.expasy.org/sprot. ‡Note that “nonessential” tyrosine can only be made from “essential”phenylanine. Nonessential Amino Acids Require Fewer Reactions for Synthesis 782 Chapter 25 Nitrogen Acquisition and Amino Acid Metabolism The predominant amino acid/␣-keto acid pair in these reactions is glutamate/ ␣-ketoglutarate, with the net effect that glutamate is the primary amino donor for the synthesis of amino acids. Transamination reactions are catalyzed by aminotransferases (the preferred name for enzymes formerly termed transami- nases). Aminotransferases are named according to their amino acid substrates, as in glutamate–aspartate aminotransferase. Aminotransferases are prime examples of enzymes that catalyze double displacement (ping-pong)–type bisubstrate reactions (see Figure 13.23). The Pathways of Amino Acid Biosynthesis Can Be Organized into Families As indicated in Table 25.1, the amino acids can be grouped into families on the basis of the metabolic intermediates that serve as their precursors. A DEEPER LOOK The Mechanism of the Aminotransferase (Transamination) Reaction The aminotransferase (transamination) reaction is a workhorse in biological systems. It provides a general means for exchange of nitrogen between amino acids and ␣-keto acids. This vital reaction is catalyzed by pyridoxal phosphate (PLP). The mechanism in- volves loss of the C ␣ proton, followed by an aldimine–ketimine tautomerization—literally a “flip-flop” of the Schiff base double bond from the pyridoxal aldehyde carbon to the ␣-carbon of the amino acid substrate. This is followed by hydrolysis of the ketimine intermediate to yield the product ␣-keto acid. Left in the active site is a pyridoxamine phosphate intermediate, which combines with another (substrate) ␣-keto acid to form a second ketimine, which rearranges to form an aldamine, followed by release as an amino acid. Transaldiminization with a lysine at the active site completes the reaction. + R C COO – H NH 3 + R C COO – N HC + CH 3 N H + 2– O 3 PO R C COO – N H 2 CH O + – N H + 2– O 3 PO R C COO – O NH 2 H 2 C O – CH 3 N H + 2– O 3 PO R' C COO – O R' C COO – N H 2 C + CH 3 N H + 2– O 3 PO + R' C COO – H NH 3 + R' C COO – H N HC + CH 3 N H + 2– O 3 PO H CH 3 H O – H + H + H 2 O H 2 O C HN 2– O 3 PO OH CH 3 N H + Lysine H O – H O – Aldimine Ketimine Ketimine Aldimine Transamination intermediate Pyridoxamine E•PLP complex E-PLP complex ᮡ The mechanism of PLP-catalyzed transamination reactions.