A study on the importance of an intact-ring structure on proline racemase catalysis

Abstract

Proline racemase (PR) catalyses the interconversion of L-proline and D-proline, in a cofactor-independent manner. PR follows a two-base mechanism, using two Cys residues in the active site that act as enantiospecific Brønsted bases, to catalyse the interconversion of the stereoisomers of proline via an aci-carboxylate intermediate. A kinetic study was conducted between the mesophilic PR from Clostridium sticklandii (CsPR) and a hyperthermophilic PR from Thermococcus litoralis (TlProR) based on their thermodynamic contributions to proline racemisation. Free energy profiles of both CsPR and TlProR suggested that they follow similar energy paths during catalysis, but CsPR showed slightly greater binding affinity with proline than TlProR. A kinetic analysis of the open-ring proline analogue, N-ethyl-L-alanine (NEA) revealed a dramatic decrease of ~4,500-fold in catalytic efficiency of CsPR-catalysed racemisation of L-NEA relative to that of L-proline.