Pyridoxamine transamination

The PEI-pyridoxamine polymer was treated with excess pyruvic acid in various buffer solutions, without added metal ions and with excess added EDTA. Kinetic studies revealed that the attached polymer increased the rate of pyridoxamine transamination with pyruvic acid by a factor of6700-8300 at pH 5.0. Under higher pH conditions, the rate enhancement decreased At pH 7.0 the rate enhancement by the polymer was still 2300 times, while at pH 8.0, the optimum for pyridoxamine itself, it was 1900. We also found that transamination by simple pyridoxamine showed strong metal ion catalysis - adding 1 equiv. of CuCl2 per pyridoxamine unit to the pH 5.0 solution increased the... 

The mechanism of the first part of transamination is shown in Figure 29.14. The process begins with reaction between the a-amino acid and pyridoxal phosphate, which is covalently bonded to the aminotransferase by an iminc linkage between the side-chain -NTI2 group of a lysine residue and the PLP aldehyde group. Deprotonation/reprotonation of the PLP-amino acid imine in steps 2 and 3 effects tautomerization of the imine C=N bond, and hydrolysis of the tautomerized imine in step 4 gives an -keto acid plus pyridoxamine... 

Hydrolysis of the a-keto acid imine by nucleophilic addition of water to the C=M bond gives the transamination products pyridoxamine phosphate (PMP) and a-keto acid. 

Pyridoxamine phosphate serves as a coenzyme of transaminases, e.g., lysyl oxidase (collagen biosynthesis), serine hydroxymethyl transferase (Cl-metabolism), S-aminolevulinate synthase (porphyrin biosynthesis), glycogen phosphoiylase (mobilization of glycogen), aspartate aminotransferase (transamination), alanine aminotransferase (transamination), kynureninase (biosynthesis of niacin), glutamate decarboxylase (biosynthesis of GABA), tyrosine decarboxylase (biosynthesis of tyramine), serine dehydratase ((3-elimination), cystathionine 3-synthase (metabolism of methionine), and cystathionine y-lyase (y-elimination). 

Figure 7-4. Ping-pong mechanism for transamination. E—CHO and E—CHjNHj represent the enzyme-pyridoxal phosphate and enzyme-pyridoxamine complexes, respectively. (Ala, alanine Pyr, pyruvate KG, a-ketoglutarate Glu, glutamate).

Be Pyridoxine, pyridoxal, pyridoxamine Coenzyme in transamination and decarboxylation of amino acids and glycogen phosphorylase role in steroid hormone action Disorders of amino acid metabolism, convulsions... 

Vitamin Ba (pyridoxine, pyridoxal, pyridoxamine) like nicotinic acid is a pyridine derivative. Its phosphorylated form is the coenzyme in enzymes that decarboxylate amino acids, e.g., tyrosine, arginine, glycine, glutamic acid, and dihydroxyphenylalanine. Vitamin B participates as coenzyme in various transaminations. It also functions in the conversion of tryptophan to nicotinic acid and amide. It is generally concerned with protein metabolism, e.g., the vitamin B8 requirement is increased in rats during increased protein intake. Vitamin B6 is also involved in the formation of unsaturated fatty acids. 

Electrophilic site for Schiff base formation with reactive amines such as a-amino acids. Corresponding transaminated species, pyridoxamine, reacts with electrophilic carbonyl compounds... 

Fig. 2. Catalytic cycle of pyridoxal/pyridoxamine-dependent transamination...

Work in the Imperiali laboratory has also focused on exploring the ability of minimal peptide scaffolds to augment the rate of coenzyme-mediated transaminations [22-25]. To accomplish this, a strategy has been developed in which the core functionality of the coenzyme is incorporated as an integral constituent of an unnatural coenzyme amino acid chimera construct. Thus, non-cova-lent binding of the coenzyme to the peptide or protein scaffold is unnecessary. Both the pyridoxal and pyridoxamine analogs have been synthesized in a form competent for Fmoc-based solid phase peptide synthesis (SPPS) (Fig. 7) [23,24]. 

The ability of peptides CBPOl-GBP 18 to modulate pyridoxamine-mediated transamination was determined by the conversion of pyruvic acid to alanine in both the absence and presence of copper(II) ion, which would be coordinated by the transamination intermediates [32]. In the absence of copper(II) ion,peptide CBP13 showed up to a 5.6-fold increase in alanine production relative to a pyridoxamine model compound and peptide CBP14 produced alanine with a 27% ee of the 1-enantiomer. In the presence of copper(II) ion, peptide CBP13 again showed the greatest increase in product production, with a 31.7-fold increase in alanine production relative to the pyridoxamine model compound. Peptide CBPIO showed optical induction for D-alanine with a 37% ee. 

The terminology vitamin Bg covers a number of structurally related compounds, including pyridoxal and pyridoxamine and their 5 -phosphates. Pyridoxal 5 -phosphate (PLP), in particular, acts as a coenzyme for a large number of important enzymic reactions, especially those involved in amino acid metabolism. We shall meet some of these in more detail later, e.g. transamination (see Section 15.6) and amino acid decarboxylation (see Section 15.7), but it is worth noting at this point that the biological role of PLP is absolutely dependent upon imine formation and hydrolysis. Vitamin Bg deficiency may lead to anaemia, weakness, eye, mouth, and nose lesions, and neurological changes. 

We have just noted the role that pyridoxal phosphate plays as a coenzyme (cofactor) in transamination reactions (see section 15.6). Pyridoxal 5 -phosphate (PLP) is crucial to a number of biochemical reactions. PLP, together with a number of closely related materials that are readily converted into PLP, e.g. pyridoxal, pyridoxine and pyridoxamine, are collectively known as vitamin Bg, which is essential for good health. 

Pyridoxal phosphate (4) is the most important coenzyme in amino acid metabolism. Its role in transamination reactions is discussed in detail on p. 178. Pyridoxal phosphate is also involved in other reactions involving amino acids, such as decarboxylations and dehydrations. The aldehyde form of pyridoxal phosphate shown here (left) is not generally found in free form. In the absence of substrates, the aldehyde group is covalently bound to the e-amino group of a lysine residue as aldimine ( Schiffs base ). Pyridoxamine phosphate (right) is an intermediate of transamination reactions. It reverts to the aldehyde form by reacting with 2-oxoacids (see p. 178). 

Among the NH2 transfer reactions, transaminations (1) are particularly important. They are catalyzed by transaminases, and occur in both catabolic and anabolic amino acid metabolism. During transamination, the amino group of an amino acid (amino acid 1) is transferred to a 2-oxoacid (oxoacid 2). From the amino acid, this produces a 2-oxo-acid (a), while from the original oxoacid, an amino acid is formed (b). The NH2 group is temporarily taken over by enzyme-bound pyridoxal phosphate (PLP see p. 106), which thus becomes pyridoxamine phosphate. 

Vitamin Bg is a mixture of six interrelated forms pyridoxine (or pyridoxol) (Figure 19.23), pyri-doxal, pyridoxamine, and their 5 -phosphates derivatives. Interconversion is possible between all forms. The active form of the vitamin is pyridoxal phosphate, which is a coenzyme correlated with the function of more than 60 enzymes involved in transamination, deamination, decarboxylation, or desulfuration reactions. 

In a number of nonenzymatic reactions catalyzed by pyridoxal, a metal ion complex is formed—a combination of a multivalent metal ion such as cupric oi aluminum ion with the Schiff base formed from the combination of an amino acid and pyridoxal (I). The electrostatic effect of the metal ion, as well as the electron sink of the pyridinium ion, facilitates the removal of an a -hydrogen atom to form the tautomeric Schiff base, II. Schiff base II is capable of a number of reactions characteristic of pyridoxal systems. Since the former asymmetric center of the amino acid has lost its asymmetry, donation of a proton to that center followed by hydrolytic cleavage of the system will result in racemic amino acid. On the other hand, donation of a proton to the benzylic carbon atom followed by hydrolytic cleavage of the system will result in a transamination reaction—that is, the amino acid will be converted to a keto acid and pyridoxal will be converted to pyridoxamine. Decarboxylation of the original amino acid can occur instead of the initial loss of a proton. In either case, a pair of electrons must be absorbed by the pyridoxal system, and in each case, the electrostatic effect of the metal ion facilitates this electron movement, as well as the subsequent hydrolytic cleavage (40, 43). 

The intermediate (32), produced upon decarboxylation of (31) may protonate at the a-carbon of the substrate (path a, Scheme 9), or at the 4 -carbon of PLP (path b). The hydrolysis of the aldimine produced by the former path accounts for the products of a-decarboxylation (equation 13). The hydrolysis of the aldimine produced in path (b) yields an aldehyde and pyridoxamine phosphate (PMP) (35). Note that this latter reaction, a decarboxylation-dependent transamination, may be classified as a redox process (reductive amination of PLP to PMP). 

In 1944, Esmond Snell reported the nonenzymatic conversion of pyridoxal into pyridoxamine (Box 14-C) by heating with glutamate. He recognized that this was also transamination and proposed that pyridoxal might be a part of a coenzyme needed for aminotransferases and that these enzymes might act via two halfreactions that interconverted pyridoxal and pyridoxamine (Eq. 14-25). The hypothesis was soon verified and the coenzyme was identified as pyridoxal 5 -phos-phate or pyridoxamine 5 -phosphate (Fig. 14-5).144/145... 

In the other, the amino acid is rotated 180° so that the a-hydrogen protrudes behind the plane of the paper. Dunathan studied pyridoxamine pyruvate aminotransferase, an enzyme closely related to PLP-requiring aminotransferases and which catalyzes the transamination of pyridoxal with L-alanine to form pyridox-amine and pyruvate. The same reaction is catalyzed by the apoenzyme of aspartate aminotransferase. In both cases, when the alanine contained 2H in the a position the 2H was transferred stereospecifically into... 

Transamination. Addition of a proton to the carbonyl carbon of the pyri-doxal leads to a compound that is the Schiff base of an a-keto acid and pyridox-amine. Hydrolysis of the Schiff base gives the a-keto acid and pyridoxamine, which may react with a different a-keto acid to reverse the sequence ... 

There is an important biochemical counterpart of the deamination reaction that utilizes pyridoxal phosphate, 7, as the aldehyde. Each step in the sequence is catalyzed by a specific enzyme. The a-amino group of the amino acid combines with 7 and is converted to a keto acid. The resulting pyridoxamine then reacts to form an imine with a different a-keto acid, resulting in formation of a new a-amino acid and regenerating 7. The overall process is shown in Equation 25-6 and is called transamination. It is a key part of the process whereby amino acids are metabolized. 

The full series of intermediates in a transamination is shown in figure 10.5a. After protonation at the aldimine carbon of pyridoxal-5 -phosphate (step 3), hydrolysis (step 4) forms an a-keto acid and pyridoxamine-5 -phosphate. The reverse of this sequence with a second a-keto acid (steps 5 through 8) completes the transamination reaction. 

In benzylamine oxidase there is evidence that the amine undergoes transamination with the pyridoxal prosthetic group to give a pyridoxamine, which is then oxidized by dioxygen to give H202 and NH3. The role for the copper is one of activation of the substrate.1346... 

Asymmetric transamination.2 This planar chiral pyridoxamine analog in the presence of Zn(C104), (l/Zn(C104)2 = 1.0.5) converts a-keto acids into (R)-amino tieids in 60 96%ee. Use of (R)-l in place of (S)-l produces (S)-amino acids with the wime elliciency. Chemical yields range from 50 75%. The preferred solvent is tnel li.mol. The pyridoxal-type analog is recovered in 75-85%yield. The transamination is considered to involve kinetically controlled stereoselective protonation of an octahedral Ztr 1 chelate intermediate. 

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