p hosphate - d ependent e nzymes
3.2 Amino a cid r acemases
d - amino acids have long been known to be important constituents of bacte-rial peptidoglycans and some antibiotics which are synthesized enzymically, rather than ribosomally. It was originally believed that the main function of mammalian d - amino acid oxidase and d - aspartate oxidase (see section 1.5.1 ) was to detoxify the small amounts of d - amino acids that may be absorbed from intestinal bacteria. More recently, free, endogenously synthesized d amino acids have been identifi ed in mammalian tissues, including the central nervous system, and d - amino acids have been identifi ed in a number of pro-teins and peptides that are synthesized on ribosomes. These may arise as a result of post - synthetic modifi cation of the protein or they may arise as a result of accidental damage to (long - lived) proteins. In aquatic crustaceans and some bivalve molluscs, d - alanine accumulates as a major osmolyte when the salinity of the environment increases.
Most of the amino acid racemases are pyridoxal phosphate - dependent, but cofactor - independent aspartate, glutamate and proline racemases have been identifi ed in archaea, bacteria and fungi, and also proline racemase in Trypanosoma cruzi , the parasite that causes Chaga ’ s disease. Diaminopimelate epimerase (see section 6.2.1 ) is also a cofactor - independent isomerase. These enzymes have a reactive cysteine residue at the active site.
Pyridoxal phosphate - dependent enzymes in bacteria include alanine race-mase (which has a low substrate specifi city and will catalyze the racemization of other amino acids as well), arginine and serine racemases. In eukaryotes, there are serine, aspartate and alanine racemases. Bacterial alanine racemase is a fold type III enzyme, while eukaryotic serine racemase is fold type II (Yoshimura & Goto, 2008 ).
In addition to amino acid racemases, bacterial d - amino acid transaminase is a signifi cant source of d - amino acids for synthesis of proteoglycan and other d - amino acid - containing peptides. d - Amino acid transaminase is also found in germinating pea seedlings (Hayashi et al ., 1990 ). d - Glutamate may be formed in two different ways. Some bacteria have glutamate racemase, while it is formed in Bacillus spp. and higher plants by a transamination reac-tion between d - alanine and 2 - oxoglutarate. In Staphylococcus spp., both pathways are used (Amadasi et al ., 2007 ).
3.2.1 Bacterial a lanine r acemase
Bacterial alanine racemase has been much studied, because of the possibility of developing specifi c inhibitors that would be clinically useful antibacterial
agents. d - amino acids are required for the synthesis of bacterial peptidogly-can; eukaryotes do not synthesize peptidoglycan. Most bacteria have two isoenzymes of alanine racemase: one is induced by d - alanine and is presum-ably mainly important in catabolism of excess d - alanine; the other is constitu-tive and is required for the production of d - alanine for peptidoglycan synthesis (Hayashi et al ., 1990 ).
The reaction of bacterial alanine racemase involves deprotonation of the α - carbon to yield a symmetrical anionic intermediate that is stabilized by formation of the pyridoxal quinonoid intermediate. This intermediate then undergoes reprotonation on the opposite side. Two separate bases are involved in the deprotonation/reprotonation reactions. They lie on opposite sides of the substrate aldimine. Tyrosine - 265 acts to deprotonate the substrate aldimine formed by l - alanine, or to reprotonate the quinonoid intermediate to yield l - alanine. Lysine - 39 acts to deprotonate the substrate aldimine formed by d - alanine, or to reprotonate the quinonoid intermediate to yield d - alanine. It is unclear how the proton is transferred between the lysyl and tyrosyl residues; it may occur via the carboxyl group of the quinonoid inter-mediate or via the phosphate group of pyridoxal phosphate (Yoshimura &
Goto, 2008 ).
3.2.2 Eukaryotic s erine r acemase
The concentration of d - serine in the mammalian brain is about one - third of that of l - serine, and higher than that of most essential amino acids. It has a clear role as a neurotransmitter or neuromodulator and is synthesized in both neurons and glia. It binds to the so called glycine binding site of the NMDA type glutamate receptor (so called because it was originally thought that the endogenous ligand for this site was glycine) and potentiates the response to glutamate.
Serine racemase knockout mice have very low brain concentrations of d serine and much reduced NMDA neurotransmission (Wolosker et al. , 2008 ).
The NMDA receptor only opens to allow calcium and sodium infl ux when both the glutamate binding site (which binds glutamate or N methyl d aspartate) and the glycine site (which binds glycine or d - serine) are occupied (Schell, 2004 ). In the silkworm ( Bombyx mori ), the activity of serine racemase and the tissue concentration of d - serine both increase considerably during metamorphosis, suggesting a morphogenic signalling role for d - serine. Silk-worm serine racemase is distinct from alanine racemase and it has only very slight activity towards alanine (Uo et al. , 1998 ).
Serine racemase has been identifi ed in fungi, plants, invertebrates and mammals, and in all species studied it also catalyzes an α – β elimination reac-tion from either l - or d - serine, yielding pyruvate, ammonium and water – the same reaction as mammalian liver l - serine deaminase (which does not have
any racemase activity and does not act on d - serine – see section 4.6.2 ). The α – β elimination and racemase activities of the enzyme are more or less equal.
In addition to pyridoxal phosphate, the enzyme also requires a divalent cation and ATP (which is an allosteric activator) for activity. There is little d - amino acid oxidase activity in the brain regions that are rich in d - serine, and theα – β elimination reaction seems to be the main way in which d - serine is catabolized in the central nervous system (Baumgart & Rodriguez - Crespo, 2008 ; De Miranda et al. , 2002 ; Foltyn et al. , 2005 ; Neidle & Dunlop, 2002 ).
Although serine racemase (which is a fold type II enzyme) has no struc-tural similarity to bacterial alanine racemase (which is a fold type III enzyme), it has a similar mechanism of action, and two bases are involved in the deprotonation/reprotonation reactions. In the enzyme from Saccharomyces pombe , these are lysine - 57 and serine - 82, which lie on opposite sides of the substrate aldimine from each other. Lysine - 57 is the residue that forms the internal aldimine with pyridoxal phosphate. Mutation of serine - 82 leads to loss of racemase and d serine deaminase activity, but it does not affect l -serine deaminase activity. This suggests that -serine - 82 is involved in the deprotonation of d - serine and the reprotonation of the quinonoid intermediate to form d serine, while lysine 57 is involved in the deprotonation of l serine and the reprotonation of the quinonoid intermediate to form l - serine (Yoshimura & Goto, 2008 ).
In rat liver serine deaminase (which does not have racemase activity) the proton that is required for the protonation of the hydroxyl group to catalyze α – β elimination is donated by the phosphate group of pyridoxal phosphate.
Elimination of theα - hydrogen and β - hydroxyl group leads to the formation of the Schiff base of pyridoxal phosphate aminoacrylate. The internal aldi-mine is then re - formed by displacement of aminoacrylate by the lysine that forms the internal aldimine. The aminoacrylate is then hydrolyzed to pyru-vate and ammonium.
In serine racemase, it is not clear whether the proton for α – β elimination comers from the phosphate group of pyridoxal phosphate, as in serine deami-nase, or whether it is the proton that has been abstracted from the α - carbon by one of the catalytic bases, lysine - 57 or serine - 82, depending on the stere-ochemistry of the substrate (Yoshimura & Goto, 2008 ).
3.2.3
D- A spartate in e ukaryotes
Just as d - serine has a role in signalling during morphogenesis in the silkworm (see previous section), d - aspartate has a role in brain development in birds and mammals. In embryonic birds and early post - natal mammals, there are transient peaks of d - aspartate that coincide with specifi c periods of develop-ment. There is an aspartate racemase in the hypothalamus, and possibly other
brain regions, and d - aspartate oxidase is widely distributed in bacteria, fungi and animals.
d - aspartate is also a neurotransmitter or neuromodulator. It is released from synaptic vesicles in response to chemical or electrical stimulation, causes an increase in neuronal concentration of cAMP and is taken up into presy-naptic neurons from the sypresy-naptic cleft by a specifi c transporter. In the hypoth-alamus, it stimulates the secretion of gonadotrophin releasing hormone and induces the synthesis of oxytocin and vasopressin mRNA. In the pituitary, it stimulates the secretion of prolactin, luteinizing hormone and growth hormone (D ’ Aniello et al. , 1993 ; Homma, 2007 ).
d aspartate is also the precursor for endogenous synthesis of N methyl d aspartate (NMDA), which is a potent agonist of the NMDA - type of gluta-mate receptor.
3.2.4
D- A mino a cids in a quatic i nvertebrates
Aquatic crustaceans and some bivalve molluscs accumulate large amounts of d - alanine in their tissues, and the amount increases as the salinity of their environment increases, suggesting that d - alanine has a role in osmoregula-tion. They have an alanine racemase that both synthesizes d - alanine as salin-ity increases and also isomerizes it to l - alanine when salinsalin-ity falls. Some species also accumulate relatively large amounts of l - glutamine, proline and glycine as osmolytes when salinity increases (Abe et al. , 2005 ).
3.2.5
D- A mino a cids in g ene - e ncoded p eptides and p roteins
The dermorphins and deltorphins in amphibian skin secretions are potent opioid hepta - peptides that have d - alanine as the second amino acid, and the deltorphins also contain d - methionine. Dermorphins are the most potent μ opioid agonists known, with a potency some 1,000 - fold higher than morphine on a molar basis. d - alanine is essential for their biological activity. Deltor-phins areδ - opioid receptor agonists.
The gene sequences show that d alanine is coded for by the normal l -alanine codon (GCG) and d - methionine by the normal codon for l methionine (AUG). The l - amino acid is incorporated on the ribosome, then, before the signal peptide sequence has been cleaved, the amino acid under-goes isomerization. This most likely occurs by deprotonation at the α - carbon, followed by reprotonation, as occurs in amino acid racemases.
There are two neuropeptides in the African giant land snail that contain d - amino acids: d - asparagine in fullicin and d - phenylalanine in achatin. Again, it is likely that these peptides are synthesized ribosomally, incorporating the
l - amino acids, followed by post - synthetic modifi cation to yield the d - amino acids (Kreil, 1997 ).
The aga toxins from the venom of the funnel web spider Agelenopsis aperta are 48 amino acid peptides that differ from each other only in that amino acid 46 may be l - or d - serine. The l - serine is slowly racemized to d - serine by a peptide isomerase that has considerable sequence homology with serine proteases (section 2.1.1 ). There is no evidence that pyridoxal phosphate is involved in the reaction, but it is likely that the reaction proceeds by way of α carbon deprotonation and reprotonation, as in the pyridoxal phosphate dependent amino acid racemases (Kreil, 1997 ).
Some d amino acids in eukaryotic proteins accumulate as a result of age related non - enzymic isomerization. d - aspartate accumulates in crystallin in the lens of the eye (a protein that apparently does not turn over throughout life) at a rate of 0.14 per cent per year. However, it can be re - isomerized to l - aspartate by way of methylation catalyzed by a protein carboxymethyl-transferase that recognizes d - aspartyl residues, followed by ester hydrolysis that involves isomerization back to l - aspartate. Two aspartyl residues in crystallin are especially sensitive to isomerization and, indeed, the ratio of d - to l - aspartyl residues may exceed 1. d - aspartyl residues also occur in amyloid plaque in the brain in Alzheimer ’ s disease, and in the aorta in athero-sclerosis (Fujii, 2005 ).
There are two groups of protein carboxymethyltransferases, both of which use S - adenosylmethionine (section 6.3.2 ) as the methyl donor (Clarke, 1985 ; Shimizu et al. , 2005 ). Type I carboxymethyltransferases catalyze the methyla-tion of specifi c aspartyl and glutamyl residues in target proteins, with a clear and consistent stoichiometry. This is a reversible post - synthetic modifi cation of target proteins that alters their function. The methylation is reversed by simple hydrolysis. In bacteria, carboxymethylation regulates the activity of chemoreceptors that control fl agellar motor activity. In the mammalian central nervous system, protein carboxymethylation is involved in the regula-tion of heterotrimeric G - proteins and protein phosphatase A, by methylating the carboxy - terminal amino acids. There is some evidence that, both in bac-teria and in mammalian central nervous systems, this methylation is impor-tant in memory (Li & Stock, 2009 ).
Type II carboxymethyltransferases catalyze the methylation of modifi ed acidic amino acid residues in proteins ( d aspartate, d glutamate and l iso aspartate), with no clear stoichiometry and no specifi city either for the site of methylation or for the proteins methylated. These enzymes are not involved in modifying the activity of their targets, but rather with labelling proteins that have undergone non - enzymic modifi cation, either as a repair mechanism or to target them for intracellular proteolysis (see section 2.1.5 ).
In iso - aspartyl residues in proteins, the α - carboxyl group is free and the peptide bond is to the β - carboxyl group. This may occur as a result of
Figure 3.4 Non - enzymic reactions leading to the formation of iso - aspartyl and D - aspartyl residues in proteins.
C H2C
H N H
C O
N H C NH2 O
C H2C
H N H
C O
N H C O -O
C H2C
H N H
C O
N C O NH3
H2O
L-asparaginyl L-aspartyl
L-succinimide
C H2C
N H
C O
N C O H
D-succinimide
C H2C
H N H
C O C
O -N H α
β
L-isoaspartyl
O H2O H2O
C H2C
N H
C O C
O -N H α
β
D-isoaspartyl O
H
H2O
C H2C
N H
C O
N H C O -O
D-aspartyl H
alignment of aspartate on tRNA, resulting in formation of a β - peptide bond on the ribosome, or as a result of non - enzymic reactions of aspartyl and asparaginyl residues in the protein, as shown in Figure 3.4 . Demethylation of methyl d - aspartyl and d - or l - iso - aspartyl residues involves dehydration to the succinimide intermediates and either hydration of l - succinimide to an l - aspartyl residue or amidation to yield an l - asparaginyl residue (Clarke, 1985 ; Shimizu et al. , 2005 ).