Aminotransferases mechanism pyridoxal phosphate

Mechanism Pyridoxal Phosphate Forms Schiff-Base Intermediates in Aminotransferases... 

FIGURE 14.22 Glutamate aspartate aminotransferase, an enzyme conforming to a double-displacement bisnbstrate mechanism. Glutamate aspartate aminotransferase is a pyridoxal phosphate-dependent enzyme. The pyridoxal serves as the —NH, acceptor from glntamate to form pyridoxamine. Pyridoxamine is then the amino donor to oxaloacetate to form asparate and regenerate the pyridoxal coenzyme form. (The pyridoxamine enzyme is the E form.)... 

Most amino acids lose their nitrogen atom by a transamination reaction in which the -NH2 group of the amino acid changes places with the keto group of ct-ketoglutarate. The products are a new a-keto acid plus glutamate. The overall process occurs in two parts, is catalyzed by aminotransferase enzymes, and involves participation of the coenzyme pyridoxal phosphate (PLP), a derivative of pyridoxine (vitamin UJ. Different aminotransferases differ in their specificity for amino acids, but the mechanism remains the same. 

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... 

In nature, aminotransferases participate in a number of metabolic pathways [4[. They catalyze the transfer of an amino group originating from an amino acid donor to a 2-ketoacid acceptor by a simple mechanism. First, an amino group from the donor is transferred to the cofactor pyridoxal phosphate with formation of a 2-keto add and an enzyme-bound pyridoxamine phosphate intermediate. Second, this intermediate transfers the amino group to the 2-keto add acceptor. The readion is reversible, shows ping-pong kinetics, and has been used industrially in the production ofamino acids [69]. It can be driven in one direction by the appropriate choice of conditions (e.g. substrate concentration). Some of the aminotransferases accept simple amines instead of amino acids as amine donors, and highly enantioselective cases have been reported [70]. 

Among the numerous enzymes that utilize pyridoxal phosphate (PLP) as cofactor, the amino acid racemases, amino acid decarboxylases (e.g., aromatic amino acids, ornithine, glutamic acid), aminotransferases (y-aminobutyrate transaminase), and a-oxamine synthases, have been the main targets in the search for fluorinated mechanism-based inhibitors. Pharmaceutical companies have played a very active role in this promising research (control of the metabolism of amino acids and neuroamines is very important at the physiological level). 

All aminotransferases have the same prosthetic group and the same reaction mechanism. The prosthetic group is pyridoxal phosphate (PLP), the coenzyme form of pyridoxine, or vitamin B6. We encountered pyridoxal phosphate in Chapter 15, as a coenzyme in the glycogen phosphorylase reaction, but its role in that reaction is not representative of its usual coenzyme function. Its primary role in cells is in the metabolism of molecules with amino groups. 

Aminotransferases operate in both directions. Their mechanism uses the cofactor pyridoxal phosphate to form Schiff bases with amino groups, as shown in Figure 4-3. 

The (I( )-l-amino-2-propanol linker is known to be derived from threonine. In S. enterica, CobD was found to be an enzyme with L-threonine 0-3-phosphate decarboxylase activity, which generates (/f)-l-amino-2-propa-nol phosphate. The enzyme is a pyridoxal phosphate requiring enzyme and the structure of the protein has been determined by X-ray crystallography (Figure 28). The structure of CobD was found to be highly similar to the aspartate aminotransferase family of enzymes. Structures of CobD with substrate and product bound have allowed a detailed mechanism for the enzyme to be proposed, whereby the external aldimine is directed toward decarboxylation rather than aminotransfer. Threonine phosphate, itself, is synthesized from L-threonine by the action of a kinase, which is encoded by pduX The pduX is housed within the propanediol utilization operon rather than the cobalamin biosynthetic operon for reasons that are not clear. 

All amino acids except lysine and threonine undergo transamination reactions. The enzymes catalyzing these reactions are known as transaminases or aminotransferases. For most of these reactions, a-ketoglutarate and glutamate serve as one of the a-keto acid-amino acid pairs. Pyridoxal phosphate is the cofactor, and the mechanism of the reaction is indicated in Figure 38.4. 

Explain the role of pyridoxal phosphate in aminotransferase reactions. Be sure to describe the Schiff base and the ketimine that are involved in the mechanism. 

Chloroalanine has been found to be an irreversible inhibitor of the pyridoxal phosphate-linked yS-aspartate decarboxylase/ aspartate aminotransferase/ and alanine racemase. The mechanism of inhibition is shown above by Eq. (7) (the sulfate reacts in the same manner) amino-ethane sulfonate irreversibly inhibits pyridoxal phosphate-linked GABA transaminase and L-serine-O-sulfate irreversibly inhibits aspartate aminotransferase. ... 

Enzymes catalyzing transfer of the a-amino group of an amino acid to the a-carbon of an a-keto acid (often a-ketoglutarate) are known as transaminases or aminotransferases. Important intermediates in such reactions are a series of Schiff bases that can be trapped by reduction with sodium borohydride (Fischer et al., 1958). These enzymes provide typical examples of double-displacement (ping-pong) mechanisms, whereby the pyridoxa-mine phosphate form is a discrete intermediate, and reaction with an a-keto acid is necessary to regenerate the pyridoxal form (Scheme 2). Although the... 

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