Glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus. Thermal denaturation and activation.
Pyrococcus furiosus is a marine hyperthermophile that grows optimally at 100 degrees C. Glutamate dehydrogenase (GDH) from P. furiosus is a hexamer of identical subunits and has an M(r) = 270,000 +/- 5500 at 25 degrees C. Electron micrographs showed that the subunit arrangement is similar to that of GDH from bovine liver (i.e. 3/2 symmetry in the form of a triangular antiprism). However, GDH from P. furiosus is inactive at temperatures below 40 degrees C and undergoes heat activation above 40 degrees C. Both NAD+ and NADP+ are utilized as cofactors. Apparently the inactive enzyme also binds cofactors, since the enzyme maintains the ability to bind to an affinity column (Cibacron blue F3GA) and is specifically eluted with NADP+. Conformational changes that accompany activation and thermal denaturation were detected by precision differential scanning microcalorimetry. Thermal denaturation starts at 110 degrees C and is completed at 118 degrees C. delta(cal) = 414 Kcal [mol GDH]-1. Tm = 113 degrees C. This increase in heat capacity indicates an extensive irreversible unfolding of the secondary structure as evidenced also by a sharp increase in absorbance at 280 nm and inactivation of the enzyme. The process of heat activation of GDH from 40 to 80 degrees C is accompanied by a much smaller increase in absorbance at 280 nm and a reversible increase in heat capacity with delta(cal) = 187 Kcal [mol GDH]-1 and Tm = 57 degrees C. This absorbance change as well as the moderate increase in heat capacity suggest that thermal activation leads to some exposure of hydrophobic groups to solvent water as the GDH structure is opened slightly. The increase in absorbance at 280 nm during activation is only 12% of that for denaturation. Overall, GDH appears to be well adapted to correspond with the growth response of P. furiosus to temperature.
Klump
H
H
Biochemistry Department, University of Cape Town, Rondebosch, South Africa.
Di Ruggiero
J
J
Kessel
M
M
Park
J B
JB
Adams
M W
MW
Robb
F T
FT
D001105:Archaea / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004789:Enzyme Activation; D005969:Glutamate Dehydrogenase / Q000737:chemistry*; D006358:Hot Temperature; D011489:Protein Denaturation; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D005969:Glutamate Dehydrogenase