Transaminases reaction mechanism

Example 8.5. Ping-pong transfer reactions. Some enzymatic transfer reactions proceed by so-called ping-pong mechanisms [40,41]. In these, the conversion of a reactant to a product leaves the enzyme in a different form. The modified enzyme then converts a second reactant to another product while itself being restored to its original form. Enzymatic transaminase reactions interconverting amino and keto acids provide a typical example [40] ... 

The detailed mechanism of pyridoxal phosphate participation in the kynureninase and kynurenine transaminase reactions is considered in detail later. Of interest in this connection is the finding that other amino... 

L-Amino acid transaminases are ubiquitous in nature and are involved, be it directly or indirectly, in the biosynthesis of most natural amino acids. All three common types of the enzyme, aspartate, aromatic, and branched chain transaminases require pyridoxal 5 -phosphate as cofactor, covalently bound to the enzyme through the formation of a Schiff base with the e-amino group of a lysine side chain. The reaction mechanism is well understood, with the enzyme shuttling between pyridoxal and pyridoxamine forms [39]. With broad substrate specificity and no requirement for external cofactor regeneration, transaminases have appropriate characteristics to function as commercial biocatalysts. The overall transformation is comprised of the transfer of an amino group from a donor, usually aspartic or glutamic acids, to an a-keto acid (Scheme 15). In most cases, the equilibrium constant is approximately 1. 

Enantiospecific reduction of C=N bonds is of interest for the synthesis of a-amino acids and derivatives such as amines. While nonenzymatic reductive amination has been known since 192711, only recently have enzymatic procedures to L-amino acids became established. The reduction can be achieved by different enzymes following different mechanisms, e.g. by pyridoxalphosphate (PLP)-dependent transaminases (E.C. 2.6.1, discussed in Chapter 12.7) or by amino acid dehydrogenases (E.C. 1.4.1) using NADH or NADPH as the cofactor. The synthetic usefulness of the transaminase reaction is diminished by the location of the equilibrium (Keq often is close... 

Longenecker and Snell (274) have proposed a sequence of reactions shown in Fig. 1 to explain nonenzymic transamination systems involving pyridoxamine and pyridoxal. The same mechanism has been proposed by these authors to account for the above observations in the case of enzymic transamination. In the latter case, the apoenzyme is considered to play the role of the metal (M in Fig. 1). While there have been some reports suggesting the requirement for a metal in enzymic transamination systems (see 273) the evidence to date is inadequate on this point. Until transaminases of very high purity are prepared this will remain a moot point. A further feature of the reaction mechanism proposed by Longenecker and Snell is that an explanation is offered for the observations by Konikova, et al. (289) and Hilton et al. (290,291) on the labilization of the a-hydrogen atom of the substrate amino acid during transamination. 

Transaminases are important enzymes in the synthesis of chiral amines, amino acids, and amino alcohols, hi this chapter the properties of transaminases, the reaction mechanisms, and their selectivity and substrate specificity are presented. The synthesis of chiral building blocks for pharmaceutically relevant substances and fine chemicals with transaminases as biocatalysts is discussed. Enzymatic asymmetric synthesis and dynamic resolution are discussed using transaminases. Protein engineering by directed evolution as well as rational design of transaminases under process condition is presented to develop efficient bioprocesses. 

Reaction mechanism of transaminases with pyridoxal-5 -phosphate (PLP) as external cofactor via a ping-pong bi-bi mechanism. A two-step reaction, starting with an internal aldimlne creating an external aldimine to pyridoxamine-5 -phosphate (PMP). 

Almost all enzymes—in contrast to the simplified description given on p. 92—have more than one substrate or product. On the other hand, it is rare for more than two substrates to be bound simultaneously. In bisubstrate reactions of the type A + B C+D, a number of reaction sequences are possible. In addition to the sequential mechanisms (see p.90), in which all substrates are bound in a specific sequence before the product is released, there are also mechanisms in which the first substrate A is bound and immediately cleaved. A part of this substrate remains bound to the enzyme, and is then transferred to the second substrate B after the first product C has been released. This is known as the ping-pong mechanism, and it is used by transaminases, for example (see p.l78). In the Lineweaver— Burk plot (right see p.92), it can be recognized in the parallel shifting of the lines when [B] is varied. 

Christen, P., and D. E. Metzler, (eds.), Transaminases. New York John Wiley and Sons, 1985. A series of review chapters describing in detail the scope and mechanisms of transamination reactions. 

Possible mechanisms of fenofibrate-induced liver injury include activation of peroxisome proliferation-activator receptors, a hypersensitivity reaction, and immune -mediated injury from cross-reactivity of the drug with autoantigens. The authors referred to six reported cases of hepatic fibrosis attributed to fenofibrate. Raised transaminase activities occur commonly with fenofibrate but are generally transient, reverse on withdrawal, and do not result in long-term injury. Fenofibrate should be withdrawn if higher than normal enzyme activities persist, and a liver biopsy should be considered if liver enzymes do not normalize after withdrawal. 

Aminotransferases (transaminases) catalyze the reversible interconversions of pairs of a-amino and a-keto acids or of terminal primary amines and the corresponding aldehydes by a shuttle mechanism in which the enzyme alternates between its PLP form and the corresponding PMP form. In the first half-reaction the PLP form of the enzyme binds the amino acid (or amine) and forms the coenzyme-substrate Schiff s base. Cleavage of the C-a—H bond is then followed by protonation at C-4. Hydrolysis of the resulting ketimine then gives a keto acid (or aldehyde), leaving the enzyme in the PMP form. The latter is recycled to the PLP form by condensation with an a-keto acid, deprotonation at C-4, protonation at C-a and transaldimina-tion to release the a-amino acid formed. 

Silverman and associates explored a variety of potential inactivators for GABA [y-aminobutyric acid, H3T (CH2)3COOH] transaminase, another pyridoxal-dependent enzyme. In the reaction of the enzyme with 4-amino-5-fluoropentanoic acid, Silverman and Invergo wrote the mechanism in equation 25 for the covalent interaction of the enzyme with the inactivator161. The mechanism, dubbed the enamine mechanism, was earlier suggested by Metzler s group162, who had also proposed, as a test, alkaline treatment of the inactivated enzyme that would result in the release of the coenzyme-bound modified inactivator. 

Induction of extrahepatic mdoleamine dioxygenase (which catalyzes the same reaction as tryptophan dioxygenase, albeit by a different mechanism) by bacterial lipopolysaccharides and mterferon-y may result in the production of relatively large amounts of kynurenine and hydroxykynurenine in tissues that lack the enzymes for onward metabolism. Kidney has kynurenine transaminase activity, and therefore extrahepatic metabolism of tryptophan may result in significant excretion of kynurenic and xanthurenic acids, even when vitamin Bg nutrition is adequate. 

These reactions are carried out by pyridoxalphosphate-dependent transaminases. Transamination reactions participate in the synthesis of most amino acids. We shall review the transaminase mechanism (Section 23.3.1) as it applies to amino acid biosynthesis (see Figure 23.10). 

Pyridoxal phosphate is a co-enzyme for numerous enzymes, notably amino acid decarboxylases, amino acid transaminases, histaminase and probably diamine oxidase Ais.iw. As most of the evidence on which the mechanism of action of pyridoxal-dependent enzymes is based has been obtained from studies of the non-enzymic interaction of pyridoxal with amino acids, these non-enzymic reactions will be considered first in some detail. 

Confirmation of the molecular structure of the enzyme-inactivator adduct has been obtained for few modified PLP-dependent enzymes. In the case of the reaction of aspartate transaminase (aspartate aminotransferase) with L-serine 0-sulfate, the surprising result thus obtained by Metzler and co-workers has forced reevaluation of the mechanism of similar inactivators (Ueno et al., 1982). Conventional wisdom argued that the reaction should involve elimination of sulfate from the inactivator followed by addition of an enzyme nucleophile to the resulting double bond (Fig. 8). When subjected to high pH, however, the inactivated enzyme releases a yellow PLP adduct which has been identified as the aldol product of the cofactor and C-3 of pyruvate (9, Fig. 9) as previously prepared by... 

Several enzymes which perform reactions at the a-carbon of amino acids are also known to catalyze an apparently unrelated exchange of the j8-hydrogens of their substrates. This capability renders glutamic-pyruvic transaminase (alanine aminotransferase) susceptible to inhibition by propargylglycine (Marcotte and Walsh, 1975), presumably by the mechanism described above. Alanine transaminase is inactivated by /3-cyano-L-alanine in an analogous manner, although... 

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