Reactions of human liver peroxisomal alanine:glyoxylate aminotransferase with beta-chloro-L-alanine and L-cysteine: spectroscopic and kinetic analysis.

Mariarita Bertoldi, Barbara Cellini, Alessandro Paiardini, Riccardo Montioli, Carla Borri Voltattorni, Mariarita Bertoldi, Barbara Cellini, Alessandro Paiardini, Riccardo Montioli, Carla Borri Voltattorni, Mariarita Bertoldi, Barbara Cellini, Alessandro Paiardini, Riccardo Montioli, Carla Borri Voltattorni

Index: Biochim. Biophys. Acta 1784(9) , 1356-62, (2008)

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Abstract

In addition to the main transaminase reaction, the pyridoxal 5'-phosphate-dependent enzyme human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is able to catalyze the alpha,beta-elimination of beta-chloro-L-alanine with a catalytic efficiency similar to that of the physiological transaminase reaction with L-alanine. On the other hand, during the reaction of AGT with L-cysteine, changes in the coenzyme forms and analysis of the products reveal the occurrence of both beta-elimination and half-transamination of L-cysteine together with the pyruvate transamination. A mechanism in which a ketimine species is the common intermediate of half-transamination and beta-elimination of L-cysteine is proposed. L-cysteine partitions between these two reactions with a ratio of approximately 2.5. Rapid scanning stopped-flow and quench flow experiments permit the identification of reaction intermediates and the measurements of the kinetic parameters of L-cysteine half-transamination. The k(cat) of this reaction is 200- or 60-fold lower than that of L-alanine and L-serine, respectively. Conversely, L-cysteine binds to AGT with a binding affinity 30- and 200-fold higher than that of L-alanine and L-serine, respectively. This appears to be consistent with the calculated interaction energies of the L-cysteine, L-alanine and L-serine docked at the active site of AGT.