p hosphate - d ependent e nzymes
3.3 Transamination
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 ).
the internal aldimine to pyridoxal phosphate is reformed, liberating the amino acid product.
Most of the transaminases are linked to one of three common metabolic intermediates as acceptor oxo - acids: pyruvate (forming alanine); oxaloace-tate (forming asparoxaloace-tate); and 2 - oxoglutarate (forming glutamate). Refer to Figures 1.9 , 1.14 and 1.15 for the roles of these transaminases in overall deamination of amino acids linked to adenosine deaminase, glutamate dehy-drogenase and glycine oxidase.
The transamination products of the amino acids are shown in Table 3.2 . Apart from lysine, all of the amino acids can undergo reversible transamina-tion; sometimes it is transamination of the oxo - acid to synthesize the amino acid, while in other cases transamination is a major route of catabolism, with the oxo - acid providing metabolic fuel or substrates for gluconeogenesis. Table 2.9 shows the metabolic fates (gluconeogenesis or ketogenesis) of amino acids in mammals, and Figure 2.2 the entry points of amino acid carbon skel-etons into the citric acid cycle. Transamination of lysine is irreversible because the oxo - acid, α - oxo - ε - aminocaproate, undergoes spontaneous dehydration and cyclization to pipecolic acid (see section 6.2.3.1 ).
The most studied transaminase is mammalian aspartate transaminase, which catalyzes transfer of the amino group between aspartate (forming oxaloacetate) and 2 - oxoglutarate (forming glutamate). The crystallized enzyme will form enzyme - substrate and enzyme - product complexes, and if the enzyme is crystallized with its substrate it undergoes a very slow reac-tion, permitting X - ray crystallography of the enzyme - substrate, enzyme intermediate and enzyme - product complexes.
Each monomer of aspartate transaminase has two domains: the coenzyme is bound to the larger domain in a pocket near the interface of the two
Figure 3.5 The reaction of transamination.
HC NH3+
COO -R1
C O COO -R1
substrate amino acid (amino donor)
substrate oxo-acid (amino acceptor)
HC NH3+ COO -R2
C O COO -R2
product amino acid
product oxo-acid
pyridoxal phosphate N H2
C HC O P O
-O -O
O OH
CH3
N H2 C
CH2
O P O
-O
-O OH
CH3 pyridoxamine phosphate
NH3+
nits; and the carboxyl groups of aspartate bind to arginine residues, one of which comes from each of the subunits. When the substrate binds, there is a conformational change in the protein. The small domain closes the active site crevice and moves one of the arginine residues, to which the substrate is bound, closer to the coenzyme in the internal aldimine. The substrate now displaces lysine - 258, which formed the internal aldimine, to form the sub-strate aldimine.
At this stage, the attachment of the coenzyme to the enzyme through the 2 ' - methyl group and the 5 ' - phosphate group is important in maintaining rigid geometry, as the coenzyme ring rotates some 30 ° about its C2 – C5 axis to bring the α - carbon of the amino acid close enough to lysine - 258 to permit this residue to catalyze the deprotonation and reprotonation to yield the ketimine.
Then, or after hydrolysis to release the oxo - acid, the coenzyme rotates back to an orientation intermediate between that in the substrate aldimine and that in the internal aldimine. The small domain then moves back to its original position, permitting release of the oxo acid product and binding of the oxo acid substrate for the second half - reaction.
In branched - chain amino acid transaminase, which is a fold type IV enzyme, the coenzyme ring rotates 180 ° about its C2 – C5 axis (Ivanov & Karpeisky, 1969 ; Karpeisky & Ivanov, 1966 ; Taylor et al. , 1990 ; Torchinsky et al. , 1990 ).
Table 3.2 Transamination products of the amino acids.
Amino acid Oxo - acid
alanine pyruvate
arginine α - keto - β - guanidoacetate aspartic acid oxaloacetate
cysteine γ - mercaptopyruvate glutamic acid 2 - oxoglutarate glutamine 2 - oxo - glutaramic acid
glycine glyoxylate
histidine imidazolepyruvate isoleucine α - keto - β - methylvalerate leucine α - keto - isocaproate [lysine (1) α - keto - ε - aminocaproate →
pipecolic acid]
methionine S - methyl - β - thiol α - oxopropionate ornithine glutamic - γ - semialdehyde
phenylalanine phenylpyruvate
proline γ - hydroxypyruvate
serine hydroxypyruvate
threonine α - keto - β - hydroxybutyrate tryptophan indolepyruvate
tyrosine p - hydroxyphenylpyruvate valine α - keto - isovalerate
1 Lysine does not normally undergo transamination; if it did, the product would undergo non - enzymic cyclization to pipecolic acid.
3.3.1 Dual s ubstrate r ecognition in t ransaminases
Transaminases have two amino acid substrates. These frequently have very different side - chains, yet the enzymes show considerable specifi city for the amino acids they bind. The two amino acid substrates have to bind to the same active site, with the α - carboxyl group in the same place (Hirotsu et al. , 2005 ).
Branched - chain amino acid transaminase (a fold type IV enzyme) has a classic ‘ lock and key ’ substrate binding mechanism. The side - chain of the amino acid is accommodated in a mainly hydrophobic pocket that will bind the hydrophobic branched side - chain, but the pocket also has three hydrophilic sites, so placed that they will recognise the γ - carboxyl group of glutamate.
The large and small domains of the enzyme do not move when substrate binds, but a small inter - domain loop moves to shield the active site from the solvent.
Aromatic amino acid transaminase (a fold type I enzyme) can bind either an aromatic amino acid or glutamate, and it has a classic induced - fi t substrate binding mechanism. There is a considerable conformational change in fold type I transaminases when the substrate binds, and the small domain moves to shield the active site from the solvent.
Both the α - carboxyl group and the side - chain bind in exactly the same places, whether the substrate is an aromatic amino acid or glutamate. However, very different active site residues surround the side chain of the substrate, depending on which substrate binds. When glutamate approaches the active site, an arginine residue from the other subunit moves to form a salt bridge with the γ - carboxyl group, and aspartate, tryptophan, serine and a water molecule are also involved in binding the side - chain. By contrast, when an aromatic amino acid approaches the active site, the conformation changes so that leucine, tryptophan and tyrosine residues form the side - chain binding site.
3.3.2 Aspartate t ransaminase and the m alate - a spartate s huttle
There are two isoenzymes of aspartate transaminase in mammalian cells: a cytosolic form and a mitochondrial form. These are involved in the malate aspartate shuttle to transfer reducing equivalents from cytosolic NADH into the mitochondrion. As shown in Figure 3.6 , the shuttle involves reduction of oxaloacetate to malate in the cytosol (with the oxidation of cytosolic NADH to NAD + ). Malate enters the mitochondria and is reduced back to oxaloac-etate, with the reduction of intra - mitochondrial NAD + to NADH. Oxaloac-etate cannot cross the mitochondrial inner membrane, but undergoes transamination to aspartate with glutamate acting as amino donor, yielding
2 - oxoglutarate, which then leaves the mitochondrion using an antiporter that transports malate inwards. Aspartate leaves the mitochondrion in exchange for glutamate entering. In the cytosol, the reverse transamination reaction occurs, forming oxaloacetate (for reduction to malate) from aspartate, and glutamate (for transport back into the mitochondrion) from 2 - oxoglutarate.
Mitochondrial aspartate transaminase is encoded by a nuclear gene; it was the fi rst such enzyme to be studied in order to determine how proteins enter the mitochondrion. Martinez - Carrion et al. (1990) cloned the gene for mito-chondrial aspartate transaminase into an expression vector and showed that the gene product was catalytically active, but differed from the purifi ed mito-chondrial enzyme in electrophoretic mobility, molecular mass and UV absorb-ance. Partial proteolysis with trypsin gave a protein that was identical to the mitochondrial enzyme, and the precursor protein was imported into isolated mitochondria. They showed that the newly synthesized protein folded into the active form, but with a hydrophobic and basic helical region exposed. This is the mitochondrial targeting sequence that is cleaved after the precursor protein has been imported into the mitochondrion.
Mitochondrial aspartate transaminase has another, quite unrelated, role in energy metabolism. The plasma membrane fatty acid binding protein that is responsible for tissue uptake of non - esterifi ed fatty acids appears to be identi-cal to mitochondrial aspartate transaminase. Mono - specifi c antisera against the proteins cross - react with each other, and the two proteins have identical electrophoretic mobility (Berk et al. , 1990 ).
Figure 3.6 The mitochondrial malate - aspartate shuttle.
Malate dehydrogenase EC 1.1.1.37, aspartate transaminase EC 2.6.1.1.
COO -C CH2
COO -O
COO -HC
CH2
COO -OH
COO -HC
CH2 COO
-NH3+
COO -HC
CH2 COO
-OH COO
-C CH2 COO
-O COO
-HC CH2
COO -NH3+
NAD+ NADH glutamate
malate dehydrogenase aspartate
transaminase
aspartate oxaloacetate malate glutamate
2-oxo-glutarate
aspartate transaminase
NAD+ NADH
malate dehydrogenase aspartate oxaloacetate malate
2-oxoglutarate