Pyridoxal Phosphate in Amino Acid Metabolism

The various reactions of pyridoxal phosphate in amino acid metabolism shown in Table 9.1 all depend on the same chemical principle - the ability to stabilize amino acid carbanions and to labilize bonds about the a-carbon, by

O" HI

N XH, lysine

N XH, pyridoxal phosphate internal aldimine (Schiff base)

NH3*

amino acid substrate lysine

decarboxylation

XHj substrate aldimine

CO, decarboxylation d

XHj substrate aldimine

R— CH2—NH3* product amine tautomerization

N CH3

lysine

N CH3

O"

I transamination

I (aminotransfer)

^N XH3 substrate ketimine

CH2NH3+ OH

^N XH3 substrate ketimine

R C COO" pyridoxamine phosphate O

product oxo-acid (keto-acid)

Figure 9.2. Reactions of pyridoxal phosphate-dependent enzymes with amino acids.

reaction of the a-amino group of the substrate with the carbonyl group of the coenzyme.

The ring nitrogen of pyridoxal phosphate exerts a strong electron withdrawing effect on the aldimine, and this leads to weakening of all three bonds about the a-carbon of the substrate. In nonenzymic reactions, all the possible pyridoxal-catalyzed reactions are observed - a-decarboxylation, aminotrans-fer, racemization and side-chain elimination, and replacement reactions. By contrast, enzymes show specificity for the reaction pathway followed; which bond is cleaved will depend on the orientation of the Schiff base relative to reactive groups of the catalytic site. As discussed in Section 9.3.1.5, reaction specificity is not complete, and a number of decarboxylases also undergo transamination.

In the absence of the amino acid substrate, pyridoxal phosphate is bound to enzymes by the formation of a Schiff base to the e-amino group of a lysine residue at the active site. As shown in Figure 9.2, the first reaction between the substrate and the coenzyme is transfer of the aldimine linkage from the e-amino group of the lysine residue to the a-amino group of the substrate.

Because pyridoxal phosphate is bound to lysine in this way, the resolution of holoenzymes to yield the apoenzyme is difficult unless this Schiff base can be reacted with a carbonyl-trapping reagent to give an adduct that can be removed by dialysis. The pyridoxamine phosphate form of aminotransferases (Section 9.3.1.3) can be resolved more readily, because the coenzyme is only held by ionic bonds to the 5' -phosphate, hydrophobic interactions with the 2 -methyl group, and hydrogen bonding to the heterocyclic nitrogen.

9.3.1.1 a-Decarboxylation of Amino Acids If the electron-withdrawing effect of the heterocyclic nitrogen of pyridoxal phosphate is primarily centered on the a -carbon-carboxyl bond, the result is decarboxylation of the amino acid aldimine and release of CO2. The resultant carbanion is then protonated, and the primary amine corresponding to the amino acid is displaced by the lysine residue at the active site, with reformation of the internal Schiff base.

A number of the products of the decarboxylation of amino acids shown in Table 9.2 are important as neurotransmitters and hormones, such as dopamine, noradrenaline, adrenaline, serotonin (5-hydroxytryptamine), histamine, and y -aminobutyric acid (GABA), and as the diamines agmatine andput-rescine and the polyamines spermidine and spermine, which are involved in the regulation of DNA metabolism. The decarboxylation of phosphatidylser-ine to phosphatidylethanolamine is important in phospholipid metabolism (Section 14.2.1).

Table 9.2 Amines Formed by Pyridoxal Phosphate-Dependent Decarboxylases Amine Parent Amino Acid Enzyme

EC No.

Agmatine

Arginine

Arginine decarboxylase

4.1

1.19

Dopamine17

DOPA

Aromatic amino acid

4.1

1.28

(dihydroxyphenylethylamine)

(3,4-dihydroxyphenylalanine)

decarboxylase

Phosphatidylethanolamine6

Phosphatidylserine

Phosphatidylserine

4.1

1.65

decarboxylase

GABA

Glutamate

Glutamate decarboxylase

4.1

1.15

Histamine

Histidine

Histidine decarboxylase

4.1

1.22

Phenylethylamine

Phenylalanine

Bacterial phenylalanine

4.1

1.53

decarboxylase

Putrescinec

Ornithine

Ornithine decarboxylase

4.1

1.17

Serotonin (5-hydroxytryptamine)

5-Hydroxy tryptophan

Aromatic amino acid

4.1

1.28

decarboxylase

Tryptamine

Tryptophan

Bacterial tryptophan

4.1

1.28

decarboxylase

Tyramine

Tyrosine

Bacterial tyrosine

4.1

1.25

decarboxylase

DOPA, dihydroxylphenylalanine; GABA, 7-aminobutyric acid.

° Dopamine is also the precursor for noradrenaline and adrenaline biosynthesis. b Phosphatidylethanolamine is the precursor for choline synthesis, Section 14.2.1.

c Putrescine is the precursor for synthesis of spermine and spermidine by reaction with decarboxylated S-adenosyl-methionine.

Pyridoxamine Glutamine Metabolism

pyridoxamine phosphate

Figure 9.3. Transamination of amino acids.

pyridoxamine phosphate

Figure 9.3. Transamination of amino acids.

9.3.1.2 Racemization of the Amino Acid Substrate Deprotonation of the a-carbon of the amino acid leads to tautomerization of the Schiff base to the quinonoid ketimine, as shown in Figure 9.2. The simplest reaction that the ketimine can undergo is reprotonation at the now symmetrical a-carbon. This is not a stereospecific process; therefore, displacement of the substrate by the reactive lysine residue results in the racemic mixture of d- and L-amino acid.

Amino acid racemases have long been known to be important in bacterial metabolism, because several D-amino acids are required for the synthesis of cell wall mucopolysaccharides. D-Serine is found in relatively large amounts in mammalian brain, where it acts as an agonist of the N-methyl-D-aspartate (NMDA) glutamate receptor. Serine racemase has been purified from rat brain and cloned from human brain (Wolosker et al., 1999; De Miranda et al., 2000).

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