Isolation and characterisation of glutamate dehydrogenase from Mycobacterium smegmatis CDC 46

Glutamate dehydrogenase (L-glutamate:NADP+ oxidoreductase (deaminating), EC 1.4.1.4) has been purified from Mycobacterium smegmatis CDC 46 using (NH4)2SO4 precipitation, negative adsorption on DEAE-cellulose, 2',5'-ADP-Sepharose affinity chromatography and Sephadex G-200. The enzyme was purified 1041.6-fold and the preparation was found to be homogeneous on column chromatography, polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. Alanine and threonine were identified as the N- and C-terminal amino acids of glutamate dehydrogenase from M. smegmastis. The enzyme kinetics and regulation of glutamate dehydrogenase activity by different nutritional factors has been studied. Initial velocity plots showed that the reaction mechanism of glutamate dehydrogenase from M. smegmatis followed an ordered sequential ter-bi mechanism.