Mechanism of racemization of amino acids by aspartate aminotransferase

Abstract

Aspartate aminotransferase (mitochondrial isoenzyme from chicken) has been found to racemize very slowly dicarboxylic amino acid substrates in the presence of their cognate oxo acids [Kochhar, S. & Christen, P. (1988) Eur. J. Biochem. 175, 433–438]. Tyrosine, phenylalanine and alanine are racemized at the same rate although they undergo the transamination reaction 3–5 orders of magnitude more slowly than the dicarboxylic substrates. Similarly, the truncated enzyme aspartate aminotransferase‐(27/32–410) catalyzes the racemization at the same rate as the native enzyme, while its rate of transamination is decreased to 3% of that of the native enzyme. Apparently, the rate‐limiting step in racemization is not immediately linked to the transamination cycle. Decreasing the water concentration in the reaction medium by adding methanol at 0°C drastically reduces the rate of racemization without affecting the rate of transamination. On the basis of these and additional kinetic data and the model of the three‐dimensional structure of the active site, we conclude that a water molecule is responsible for the protonation of Cα of the coenzyme‐substrate intermediate from the wrong side. The diffusion of the water molecule into the interior of the enzyme appears to be the rate‐limiting step in aspartate‐aminotransferase‐catalyzed racemization. Copyright © 1992, Wiley Blackwell. All rights reserved