Regulatory properties of glutamate dehydrogenase from Sulfolobus solfataricus

The purified glutamate dehydrogenase (GDH) from Sulfolobus solfataricus sho wed remarkable thermostability and retained 90-95% of the initial activity after incubation at -20 degrees C, 4 degrees C, and 25 degrees C for up to 6 months. Unlike mammalian GDHs, the activity of GDH from Sulfolobus solfat aricus was not significantly affected by the presence of various allosteric effecters such as ADP, GTP, and leucine, Incubation of GDH with increasing concentration of o-phthalaldehyde resulted in a progressive decrease in en zyme activity, suggesting that the o-phthalaldehyde-modified lysine or cyst eine is directly involved in catalysis, The inhibition was competitive with respect to both 2-oxoglutarate (K-i = 30 mu M) and NADH (K-i = 100 mu M), further supporting a possibility that the o-phthalaldehyde-modified residue s may be directly involved at the catalytic site. The modification of GDH b y the arginine-specific dicarbonyl reagent phenylglyoxal was also examined with the view that arginine residues might play a general role in the bindi ng of coenzyme throughout the family of pyridine nucleotide-dependent dehyd ro-genases, The purified GDH was inactivated in a dose-dependent manner by phenylglyoxal, Either NADH or 2-oxoglutarate did not gave any protection ag ainst the inactivation caused by a phenylglyoxal, This result indicates tha t GDH saturated with NADH or 2-oxoglutarate is still open to attack by phen ylglyoxal, Phenylglyoxal was an uncompetitive inhibitor (K-i = 5 mu M) with respect to 2-oxoglutarate and a noncompetitive inhibitor (K-i = 6 mu M) wi th respect to NADH. The above results suggests that the phenylglyoxal-modif ied arginine residues are not located at the catalytic site and the inactiv ation of GDH by phenylglyoxal might be due to a steric hindrance or a confo rmational change affected by the interaction of the enzyme with its inhibit or.