CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate
Subject Chemistry
Paper No and Title Paper 16, Bioorganic and biophysical chemistry Module No and Title Module 27, Co-enzyme-III pyridoxal phosphate
Module Tag CHE_P16_M27
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate
TABLE OF CONTENTS
1. Learning outcomes 2. Introduction
2.1 Non-enzymatic models
3. Variety of PLP dependent reaction 3.1 By loss of α - hydrogen
3.2 By removal of α-carboxylate or decarboxylation 3.3 Reactions by side chain cleavage
3.4 Reactions of ketimine intermediate as e- acceptor 4. Pyrodoxamine phosphate as a Coenzyme
5. Stereochemistry of PLP- requiring enzymes
6. Summary
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate 1. Learning Outcomes
After studying this module, you shall be able to
Know about the chemical structure of pyridoxal phosphate.
Learn about various reactions carried out by different enzymes using pyridoxal phosphate as coenzyme.
Understand pyridoxal phosphate as co-enzyme in various reactions such as transamination, decarboxylation, non-oxidative deamination, transulfuration and condensation.
2. Introduction
The pyridoxal phosphate is a phosphate ester of aldehyde form of vitamin B6. Pyridoxal phosphate [or PLP]. Vitamin B6, which is also known as anti-dermatitis factor includes three closely related forms, pyridoxanine, pyridoxal and pyridoxine.
Fig1. Structure of pyridoxine, pyridoxal and pyridoxamine
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate
Active forms are phosphate ester such as pyridoxal phosphate and pyridoxamine phosphate or coenzyme form are formed by phosphorylation formed by enzyme pyridoxal kinase using ATP.
Fig2. Structure of pyridoxal phosphate and pyridoxamine phosphate
Pyridoxal phosphate is required by many enzymes catalysing reactions of amino acid and amines.
The reactions are numerous and pyridoxal phosphate is surely on of nature's most versatile catalyst. The story begins with biochemical transamination. In 1937, Alexander Braunstein and Maria Kritzmann, in Moscow, described the transamination reaction by which amino groups can be transferred from one carbon skeleton to another. The transamination reaction is a widespread process of importance in many aspects of nitrogen metabolism of organisms. For large number of transaminases glutamate is one of the reactant.
In 1944, Esmond snell reported the non-enzymatic conversion of pyridoxal into pyridoxamine by heating with glutamate. He recognized that this was also trannsamination and proposed that pyridoxal might be a part of coenzyme needed for amino transterases. The hypothesis was soon verified and coenzyme was identified as pyridoxal 5’-phosphate or pyridoxamine 5’- phosphate.
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate
2.1 Non-enzymatic models
Pyridoxal or PLP, in complete absence of enzymes, not only goes slow transamination with amino acids but also catalysis many other reactions of amino acids that are identical to those catalyzed by PLP-dependent enzymes. Thus, the coenzyme itself can be regarded as the active site of enzymes and can be studied in non-enzymatic reactions. From such studies, Snell and associates drew following conclusion.
(a) The aldehyde group of PLP reacts readily and reversibly with amino acid to form schiff
bases which react further to give product.
Fig3. Pyridoxal phosphate in Schiff base linkage to enzyme lysine residue (
b) For an aldehyde to be a catalyst, a strong electron attracting group e.g. the ring nitrogen of pyridine as in (PLP) must be ortho or para to CHO.3. The variety of PLP dependent reactions
The reaction shown in general mechanism of action of PLP fall into four groups
3.1 Group A: By loss of α-hydrogen
Dissociation of α- hydrogen from schiff base leads to a guinoide-carbanionic intermediate the name reflects the characterization of two resonance forms drawn. This intermediate ion can react in several ways
i. Racemization ii. Transamination
iii. Beta elimination or replacement
Amino acid transformation at α-carbon that are facilated by pyridoxal phosphate
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate
3.1.1 Racemization
A proton can be added back to the original α position but without stereospecifity. A racemase which does this is important to bacteria. They must synthesize D-alanine and D-glutamic acid from corresponding L-isomers for use in formation of their peptide glycan envelope.
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate
3.1.2 Transamination of Amino Acids
Amino transferases are classic examples of enzymes biomolecular ping-pong reactions in which first substrate reacts and product must leave the active site before the second substrate can bind.
Thus the incoming amino acid binds to active site, donates its amino group to pyridoxal phosphate and departs in the form of a α-keto acid. The incoming α-keto acid then binds, accepts the amino group from pyridoxamine phosphate and departs in the form of amino acid.
Most amino acid undergo transamination reaction with few exception like lysine, threonine etc.
Mechanism of transamination showing the role of pyridoxal phosphate as coenzyme.
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate
Fig4. Mechanism of transamination showing the role of pyridoxal phosphate as coenzyme.
3.1.3 Elimination and β replacement (non-oxidative deamination)
When a good leaving group is present in the β position of amino acid it can be eliminated. A large number of such enzymes are serine and threonine dehydratases, which eliminate OH- and H2O.
Reaction Mechanism of serine dehydratase
3.2 Group B: Removal of α-carboxylate or decarboxylate
Several amino acids undergo decarboxylation requiring B6 as a coenzyme. Some important examples of decarboxylation are mentioned:
5- hydroxytryptophan to serotonine
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate
Histidine to histamine
Glutamate to GABA and
DOPA to dopamine
Role of Vitamin B6 in amino acid decarboxylation reaction
The bond to the carboxyl group of amino acid substrate is broken in reaction catalyzed by amino acid decarboxylases. These also presumbably lead to transient guinonoid carbanionic intermediate. Addition of proton at the original site of decorboxylation followed by breakup of schiff base completes the sequence. Decarboxylation of amino acid is nearly irreversible and frequently appears as a final step in synthesis of amino acid compounds.
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate
3.3 Group C: Side chain cleavage (Condensation reactions of amino acid)
In this type a reaction of side chain of schiff base undergoes aldol cleavage. Conversely, a side chain can be added by β condensation. The best known enzyme of this group is serine hydroxymethyl transferase which converts serine to glycine and formaldehyde. The latter is not released in a free form but is transferred by same enzyme specifically to tetrahydro folic acid.
Threonine is cleaved to acetaldehyde by same enzyme
Reaction mechanism of serine hydroxymethyl transferase
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate
In the same group ester condensation reactions join acyl group form CoA derivatives to schiff bases derived from glycine or serine. Succinyl CoA is the acyl donor for biosynthesis of δ-amine levulinic acid, an intermediate in porphyrin synthesis.
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate
The enzyme does not catalyze decarboxylation of glycine in absence of succinyl CoA.
A similar reaction in biosynthesis of sphingosine serine is condensed with palmitoyl CoA an amino ketone intermediate.
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate
3.4 Group D: Reaction of ketamine intermediate as electron acceptor
The fourth group of PLP-dependent reactions are thought to depend upon formation of the ketimine intermediate. In this form the original α hydrogen of the amino acid has been removed and the C=NH+ bond of the ketimine is polarized in a direction that favors electron withdrawal from the amino acid into the imine group. This permits another series of enzymatic reactions analogous to those of the β-oxo acid. Both elimination and C-C bond cleavage α,β to the C=N group of the ketimine can occur.
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate
Enzymes of this group catalyze elimination of γ substituent from amino acids. Eliminated groups may be replaced by other substituent, either in the α or the β positions. The ketimine formed initially by such an enzyme undergoes elimination of the γ substituent (β with respect to the C=N group) along with a proton from the β position of the original amino acid to form an unsaturated intermediate which can react in one of three ways, depending upon the enzyme. Addition of HY’
leads to γ replacement, while addition of a proton at the α position leads to an α β-unsaturated schiff base. The latter can react by addition HY’ or it can break down to an α- oxo acid and ammonium ion, just as in the β elimination reactions discussed earlier. An important γ replacement reaction is conversion of O-acetyl-, O-succinyl-, or O-phosphohomoserine to cystathionine. This cystathionine γ - synthase reaction lies on the pathway of biosynthesis of methionine by bacteria, fungi, and higher plants. Subsequent reactions include B elimination from cystathionine of homocysteine which is then converted to methionine.
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry
MODULE No.27 Co-enzyme-III pyridoxal phosphate
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry
MODULE No.27 Co-enzyme-III pyridoxal phosphate
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate
Mechanism of Transsulfuration
Glycogen phosphorylase: While PLP is ideally designed to catalyze reactions of amino compounds it was surprising to find it as an essential cofactor for glycogen phosphorylase. The PLP is linked as Schiff base in the same way as in other PLP-dependent enzymes, but there is no obvious function for the coenzyme ring. The phosphate group probably acts as an acid-base catalyst. It has been estimated that 50% of the vitamin B6 in our body is present as PLP in muscle phosphorylase. Studies of vitamin B6- deficient rats suggest that PLP in phosphorylase serves as a reserve supply, much of which can be taken for other purposes during times of deficiency.
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate 4. Pyridoxamine phosphate as a Coenzyme
If PLP is a cofactor designed to react with amino groups of substrates, might not pyridoxamine phosphate (PMP) act as a coenzyme for reactions of carbonyl compounds? An example of this kind of function has been found in the formation of 3,6 dideoxyhexoses needed for bacterial cell surface antigens. Glucose (as cytidine diphosphate glucose CDP glucose) is first converted to 4- oxo-6-deoxy-CDP-glucose. The conversion of the latter to 3,6-dideoxy-CDP-glucose requires PMP as well as NADH or NADPH.
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate 5. Stereochemistry of PLP-Requiring Enzymes
According to stereoelectronic principles, the bond in the substrate amino acid that is to be broken by a PLP-dependent enzyme should lie in a plane perpendicular to the plane of the cofactor-imine π system. This would minimize the energy of the transition state by allowing maximum σ-π overlap between the breaking bond and the ring- imine system. It also would provide the geometry closest to that of the planar quinonoid intermediate to be formed, thus minimizing molecular motion in the approach to the transition state.
Another related reactions that goes through a ketamine is conversion of amino acid kynurenine to alanine and anthranilic acid: [Tryptophan metabolism]
Vit B6 as Co-enzyme in tryptophan metabolism:
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate
6. Summary
Pyridoxal phosphate (PLP) is the active form of vit B6.
PLP is formed from phosphorylation of all three form of vitamin B6.
Vitamin B6 consists of a mixture of three different closely related pyridine derivatives such as Pyridoxine, Pyridoxal and Pyridoxamine.
CHEMISTRY PAPER No. 16 Bioorganic and Biophysical Chemistry MODULE No.27 Co-enzyme-III pyridoxal phosphate
Pyridoxal phosphate acts as coenzyme in large number of reactions of amino acid metabolism.
Some involve removal of l- hydrogen, in this group transmination, racemisation and deaminases come.
Second group involve removal of l-carboxylate as Co2 which involve all decarboxylase like DOPA decarboxylate ornithine decarboxdylase, Histidine decarboxylase and many more.
Third group involve removal or replacement of side chain (or -H) by aldol cleave; which involve serine hydroxy methyl transferase, threonine aldolase etc.
Fouth group involve reaction of ketimine intermediates involving cystathione γ -synthase, anthranilic acid synthese etc. other PLP-dependent reactions.
Pyridoxal phosphate is required for niacin coenzyme (NAD+/NADP+) synthesis from tryptophan
The enzyme glycogen phosphorylase contain covalently bound PLP
