NAD-specific glutamate dehydrogenase fromThermus thermophilusHB8: purification and enzymatic properties

Abstract

An NAD-specific glutamate dehydrogenase from Thermus thermophilus HB8 was purified 350-fold by a several-step procedure involving Blue-Sepharose chromatography. The native protein had a molecular mass of approximately 289 kDa, and consisted of six subunits with a molecular mass of 48 kDa each. The optimum pH for the deaminating reaction was 8.0. The optimum temperature was around 85–90°C. NAD-glutamate dehydrogenase showed a high coenzyme specificity, catalysed preferentially glutamate catabolism and presented Km values for NAD and l-glutamate of 0.27±0.03 mM and 49±10 mM, respectively. No activity was detected with NADH or NADPH, 2-oxoglutarate and ammonia. Enzyme activity was found to be very stable at 72°C. Differential scanning calorimetry revealed a tm of 95°C for denaturation.