Aminotransferase half reactions

From the sequence of reactions found it follows that copper-quinoprotein amine oxidases catalyze an aminotransferase reaction. A different reaction sequence occurs with flavoprotein amine oxidases (EC 1.4.3.4), where formation of NH3 is not dependent on the presence of 02. However, since reductive trapping of amines in the first half-reaction [86] showed attachment of substrate but not of tritium, the mechanism is also different from the aminotransferase reaction that... 

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. 

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 experiments conclusively prove that the addition of hydrogen to C-4 of the coenzyme occurs at the Si face of the Schiff base. Evidence has already been provided for the syn nature of the tautomeric process in the reaction catalysed by pyridoxamine-pyruvate aminotransferase [107]. If the same precedent is extended to aspartate aminotransferase it then follows that the bond to C that is formed and broken in this case must also be located on the Si face at C-4, in the catalytic complex, as shown in structure 2 (Fig. 53). In other words, the alternative arrangement for syn proton transfer shown in 1 (Fig. 53) is precluded by these experiments. The direction of hydrogen addition to C-4 of the coenzyme in the half-reaction has also been studied using several other L-amino add requiring aminotransferases and in every case the medium hydrogen was shown to add to the Si face at C-4 (Table 5). These experiments have led to the generaUsed view that in B -dependent reactions... 

Figure 14-10 Models of catalytic intermediates for aspartate aminotransferase in a half-transamination reaction from aspartate to oxalocetate. For clarity, only a selection of the active site groups are shown. (A) Michaelis complex of PLP enzyme with aspartate. (B) Geminal diamine. (C) Ketimine intermediate. The circle indicates a bound water molecule. See Jansonius and Vincent in Jurnak and McPherson.163 Courtesy of J.N. Jansonius.

Answer The second amino group introduced into urea is transferred from aspartate. This amino acid is generated in large quantities by transamination between oxaloacetate and glutamate (and many other amino acids), catalyzed by aspartate aminotransferase. Approximately one half of all the amino groups excreted as urea must pass through the aspartate aminotransferase reaction, and liver contains higher levels of this aminotransferase than of any other. 

The reactions catalyzed by aminotransferases arc called transaminahon reactions. It might he not that in these reactions the amino group being transferred initially is transferred to the cofactor pyridoxal phosphate, resulting in its conversion to pyridoxamine phosphate. In the second half of the reaction, the amino group residing on the cofactor is transferred to the keto acid cosubslrate, thus regenerating the cofactor in the pyridoxal phosphate form. As stated earlier, the cofactor remains bo Lind to the enzyme when it occurs as the pyridoxal phosphate and pyridoxamine phosphate forms. 

A bacterial aminotransferase [46] promotes a decarboxylative transamination reaction with a-aminoisobutyrate in the presence of pyruvate. The reaction occurs via the sequence of Fig. 12 involving an initial cleavage of the Q-COjH bond in the substrate pyridoxal-P Schiff base complex (Fig. 12, 1) followed by reprotonation at C-4 of the coenzyme to give the pyridoxamine-P-enzyme complex (Fig. 12, 4) that participates in the transamination of pyruvate. However, the enzyme will also transform L-alanine at a significant rate by a half-transamination reaction into pyruvate, thereby implying that it is now the C -H bond of the amino acid that is first broken. 

Fig. 44. Half-transamination reaction catalysed by carbamylated aspartate aminotransferase.

Fig. 48. Chirality analysis at C-4 of pyridoxamine obtained following reduction of the binary complex of aspartate aminotransferase with NaB H4. It should be noted that at the dehydrohalogenation step only half the reaction will occur via the pathway shown (cf. reaction of 4 Fig. 49).

The pattern recorded above raises the question whether the change of face at C-4 to the solvent side is a mandatory requirement in the transformation of binary into ternary complexes in pyridoxal-P-dependent reactions. That this may be so was the view beginning to prevail until a timely reminder, or perhaps an undue caution, came from a more recent report by Zito and Martinez-Carrion [93]. As has already been cited, these workers repeated the earlier experiments of the Zurich School on aspartate aminotransferase confirming the Re face hydride attack at C-4 in the binary complex. However, aspartate aminotransferase carbamylated at the active site Lys-258 was used to produce the substrate-coenzyme Schiff base linkage in the ternary complex. Since the modified enzyme catalysed the half-transamination reaction ... 

Fig. 55. Configurational analysis of [4 - H]pyridoxamine obtained by the half-transamination reaction catalysed by the apo protein of aspartate aminotransferase in [ H]H20.

The half-life of pyruvic acid in the presence of an aminotransferase enzyme (which converts it to alanine) was found to be 221 s. How long will it take for the concentration of pyruvic acid to fall to of its initial value in this first-order reaction ... 

Enzymes present in the liver cytosol with short half-lives include ornithine decarboxylase, thymidine kinase, tyrosine aminotransferase, tryptophan oxygenase, hydroxymethylglutaryl-CoA reductase, serine dehydratase, and phosphoenolpyruvate carboxykinase. All of these enzymes have degradation rate constants greater than 0.1/h—more than 10 times more rapid than the average ka for liver cytosol proteins (Schimke, 1970). Perhaps a scrutiny of the group can provide information on the enzyme properties as well as the nature of reactions catalyzed by enzymes with rapid turnover rates. 

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