Aspartate aminotransferase kinetics

Aspartate aminotransferase catalyses the transfer of the NH2 group between aspartic acid and 2-oxoacids via the intervention of a pyridoxal coenzyme. The kinetically significant step, a 1,3-proton transfer, is thought to involve lysine-258 in a proton relay system (Scheme 18) replacement of lysine-258 with an alanine residue by site-directed muta-tion Q yields a mutant enzyme which is only weakly active but which can have activity restored by added amines (Scheme 18). 

In the case of reaction of aspartate aminotransferase with erythro- -hydroxy-aspartate, a total of 8 relaxation processes were resolved by Hammes and Haslam [2]. Steady-state kinetics could not have detected such isomerisations of an enzyme-substrate complex as they do not involve reaction with any additional ligand. 

The enzyme can be measured in a kinetic manner by a coupled enzyme reaction system. The oxaloacetate formed by aspartate aminotransferase is converted to malate by including malate dehydrogenase in the assay system. This is accompanied by the oxidation of NADH to NAD which can be followed spectrophotometrically at 340 nm. 

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