CHARACTERIZATION OF L-GLUTAMINE-D-FRUCTOSE-6-PHOSPHATE AMIDOTRANSFERASE FROM AN EXTREME THERMOPHILE THERMUS-THERMOPHILUS HB8

Glucosamine-6-phosphate synthase from the extremophile Thermus thermop hilus (GlmS(th)) was purified to homogeneity from an Escherichia coli overproducer. The homodimeric enzyme exhibits an optimum activity at 7 0 degrees C with a half-life of 90 min at 80 degrees C, Dissociation e xperiments in guanidinium chloride and urea are consistent with the ab sence of catalytic activity of the monomer, Differential scanning micr ocalorimetry analysis of GlmS(th) revealed an irreversible denaturatio n process with a Delta H-cal = 257 kcal . mol(-1) and T-m = 82,6 degre es C, Antigenic cross-reaction with GlmS(th) was observed with the E. coli enzyme using monoclonal antibodies (mAbs) specific for linear epi topes of the glutamine binding domain, However, no cross-reactivity wa s observed with an mAb specific for a native conformation of the E, co li enzyme, The inhibition constants of 6-diazo-5-oxo-L-norleucine and methoxyfumaroyl-L-2,3-diaminopropionic acid, potent glutamine site dir ected affinity labels of the E, coli enzyme, were reduced by 2 to 3 or ders of magnitude when tested on GlmS(th), whereas the properties of 2 -amino-2-deoxyglucitol-6P, a potent competitive inhibitor of the fruct ose-6P site, remained unaffected, These data, combined with its unexpe cted resistance to limited proteolysis, are consistent with an increas e in the structural constraint of the thermophile enzyme vs its mesoph ilic. counterpart, (C) 1997 Academic Press, Inc.