Steps in the Transaminase Reaction

4 Steps in the Transaminase Reaction Purified aspartate aminotransferase is capable of catalyzing the half-reaction of transamination (very slowly) in the crystal. This means that the conformational changes that occur during the reaction can be followed by X-ray diffraction crystallography. 

The deprotonated aldimine is reprotonated at carbon-4 by reaction with a histamine residue to form the pyridoxamine phosphate ketimine. Hydrolysis of this complex yields the free oxoacid (oxaloacetate), leaving pyridoxamine phosphate at the catalytic site (Ivanov and Karpeisky, 1969). 

5 Tyansamination Reactions of Other Pyridoxal Phosphate Enzymes In addition to their main reactions, a number of pyridoxal phosphate-dependent enzymes also catalyze the half-reaction of trcuisamination. Such enzymes include serine hydroxymethyltransferase (Section 10.3.1.1), several deccuboxylases, emd kynureninase (Section 8.3.3.2). 

The result of this hedf-transaminEise reaction is formation of pyridoxamine phosphate at the active site of the enzyme, euid hence loss of activity. Pyridoxamine phosphate dissociates from the active site, so that if adequate pyridoxal phosphate is available the resultant apoenzyme can be reactivated. 

Theratio oftrtmstuninatiomdecEuboxjdationisrelativelysmEill-ofthe order of 1 10,000 for glutamate decarboxylase. Nevertheless, this is sufficient to result in significemt loss of active enzyme, and Meister (1990) su ested that this may be a control mechanism rather them simply a lack of reaction specificity. 

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The experiments described earlier showed that in liver homogenates and extracts this reaction is brought about by transamination, which is an obligatory first step in the oxidation of tyrosine by such systems. The existence of su( h a transaminating system was already known (133, 134, 393), and the observed pyridoxal phosphate-dependence when transamination was was made rate-controlling (489) was in accordance with the known behavior of transaminases (c/. 482). 

The first step in the catabolism of most amino acids is the transfer of the o-amino group from the amino acid to a-ketoglutarate (tx-KG). This process is catalyzed by transaminase (aminotransferase) enzymes that require pyridoxal phosphate as a cofactor. The products of this reaction are glutamate (Glu) and the a-ketoacid analog of the amino acid destined for catabolic breakdown. For example, aspartate is converted to its a-keto analog, oxalo-acetate, by the action of aspartate transaminase (AST), which also produces Glu from a-KG. The transamination process is freely reversible, and the direction in which the reaction proceeds is dependent on the concentrations of the reactants and products. These reactions do not effect a net removal of amino nitrogen the amino group is only transferred from one amino acid to another. 

In the described examples, the pyridoxamine was covalently attached to the polymer while in most real transaminase enzymes the pyridoxamine coenzyme forms a noncovalent active holoenzyme with the protein (apoenzyme). A new artificial transaminase mimic was developed, in which the pyridoxamine binds noncovalently and reversibly to the polymer. The pyridoxamine attached, for example, to a steroid side chain 99 or 100, together with modified PEI 101 (molecular weight of 60000 and 8.7% dodecyl chains) forms the artificial holoenzyme (Figure 38a). The transamination of pyruvic acid was accelerated 28000-fold with 99 + 101 compared to 10 000 with the covalent pyridoxamine-polymer 98 enzyme mimic. This was due to the fact that the noncovalent system 99 - -101 is more dynamic and therefore can adopt a more suitable geometry for the reaction. The artificial transaminase shows effective rate enhancements in converting the ketoacid into the amino acid, but also the pyridoxamine is converted to pyridoxal. The conversion to pyridoxamine is a necessary step in the catalytic cycle to achieve high turnovers however, this was still not possible with the noncovalent model system. It was observed that the reverse process is very slow and actually in all artificial models so far thermodynamically unfavorable. However, it was possible to use sacrificial amino acids at elevated temperatures (60 °C) that were decarboxy-lated to recycle the pyridoxal 102 to pyridoxamine 100 with modest turnover numbers of 81 (Figure 38b). " ... 

The alanine racemization catalyzed by alanine racemase is considered to be initiated by the transaldimination (Fig. 8.5).26) In this step, PLP bound to the active-site lysine residue forms the external Schiff base with a substrate alanine (Fig. 8.5, 1). The following a-proton abstraction produces the resonance-stabilized carbanion intermediates (Fig. 8.5, 2). If the reprotonation occurs on the opposite face of the substrate-PLP complex on which the proton-abstraction proceeds, the antipodal aldimine is formed (Fig. 8.5,3). The subsequent hydrolysis of the aldimine complex gives the isomerized alanine and PLP-form racemase. The random return of hydrogen to the carbanion intermediate is the distinguishing feature that differentiates racemization from reactions catalyzed by other pyridoxal enzymes such as transaminases. Transaminases catalyze the transfer of amino group between amino acid and keto acid, and the reaction is initiated by the transaldimination, followed by the a-proton abstraction from the substrate-PLP aldimine to form a resonance-stabilized carbanion. This step is common to racemases and transaminases. However, in the transamination the abstracted proton is then tranferred to C4 carbon of PLP in a highly stereospecific manner The re-protonation occurs on the same face of the PLP-substrate aldimine on which the a-proton is abstracted. With only a few exceptions,27,28) each step of pyridoxal enzymes-catalyzed reaction proceeds on only one side of the planar PLP-substrate complex. However, in the amino acid racemase... 

E-11) SCOT (serum glutamate oxaloacetate transaminase), more recently called AST (aspartate aminotransferase), acts at this step. Both names make sense, depending on which way you read the chemical reaction. The enzyme is found in many areas of the body, but Is most useful as a marker of hver or cardiac injury. It leaks out of the damaged cell and increases in the smim after myocardial infarction and liver injury (for instance, hepatitis) and may pros ids clua as to the existence cf theeo coiuhtions. 

Acylphosphonic acids react with pyridoxamine, which is a coenzyme of transaminases, with the formation of a-aminophosphonic acids (see also reductive amination. Section II. C. 4. c) The first step of the reaction is addition of the pyridoxamine to the carbonyl of the acylphosphonate, followed by prototropic rearrangement and hydrolysis to pyridoxal and an aminophosphonic acid (equation 59). This reaction is in contrast with the reverse... 

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