L-Alanine Glyoxylate Aminotransferase

Located in the peroxisomes of liver, L-alanine-glyoxylate aminotransferase catalyzes the transamination of alanine and glyoxylate to form pyruvate and glycine. A rare inborn error of metabolism manifested as hyperoxaluria is due to a deficiency of this enzyme. 

The phenylhydrazone derivative of pyruvate is quantitated by chromatography on a LiChrospher RP-8 column (4 mm x 250 mm, 5 /tm). Isocratic elution was performed at room temperature and a flow rate of 1.0 mL/min by using a ternary mixture of 14.7 mM KH2P04, 8.76 mM K2HP04, and HPLC-grade methanol (75 10 15, v/v/v). The eluate was monitored at 314 nm. 

The reaction mixture contained 10 mM glyoxylate, 80 mM L-alanine, 100 mM potassium phosphate buffer (pH 8.0), and 100 fiM pyridoxal-5 -phosphate in a final volume of 281 fiL. The reaction was initiated by adding alanine. After 30 minutes the reaction was stopped by mixing a 50 fiL aliquot of the incubated sample with 50 fiL of phenylhydrazine solution (1M in water) and 2.0 mL of water. After 15 minutes of incubation at room temperature, 50 fiL of the mixture was used for analysis by HPLC. 

The assay was used to determine activity in liver specimens obtained by biopsy. Samples were homogenized by sonication in a solution containing 0.1 M potassium phosphate (pH 8.0) and 0.25 M sucrose. 

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One case where enzymatic involvement is documented is that of (R)-a-fluoro-/3-alanine (11.42), itself the major (>80%) metabolite of 5-fluoroura-cil (11.16) in humans. With rat liver homogenates, it was demonstrated that mitochondrial L-alanine-glyoxylate aminotransferase II (AlaAT-II, EC 2.6.1.44) catalyzed the defluorination of (R)- and (S)-a-fluoro-/3-alanine with catalytic efficiencies of 0.038 and 0.050 mM"1 s 1 at 37° and pH 7.0, respectively, [76], The primary product of the reaction was jS-aminoacrylate... 

Amination of aldehydes by an amino acid aldehyde aminotransferase from Mercurialis perennis was inhibited strongly by pyruvate, oxaloacetate, and oxoglutarate, and this inhibition was suspected to be competitive (Hartmann et al., 1972). Formation of coniceine by an alanine 5-keto-octanal aminotransferase was inhibited competitively by pyruvate and uncompetitively by glyoxylate (Roberts, 1978). With the L-ornithine 2-oxoacid aminotransferase from Cucurbita pepo, severe inhibition by valine, leucine, and isoleucine could be observed, but there was no inhibition by lysine or proline (Lu and Mazelis, 1975). Cucurbita maxima ornithine aminotransferase, however, was reported to be inhibited by proline (Splittstoesser and Fowden, 1973), as well as by canavanine and diaminobutyrate. 

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