PURIFICATION AND CHARACTERIZATION OF GLUCOSE-6-PHOSPHATE-DEHYDROGENASE FROM CRYPTOCOCCUS-NEOFORMANS - IDENTIFICATION AS NOTHING DEHYDROGENASE

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was purified from Cryp tococcus neoformans, a basidiomyceteous yeast that is an opportunistic pathogen of AIDS patients. The enzyme had a subunit molecular weight of 5 X 10(4), a specific activity of 50 units mg(-1), and K-m values f or NADP and glucose-6-phosphate of 1.6 and 24 mu M, respectively. The enzyme catalyzed the dehydrogenation of glucose, in the presence of di methylsulfoxide, with K-m of 5 mM and V-max 10% of that with glucose-6 -phosphate. pH profiles indicated the presence of a group with pK(a) o f 6.6 that is involved in catalysis, and groups with pK(a)s of about 8 .8 that are involved in binding of NADP and glucose-6-phosphate. The e nzyme was inhibited by NADPH, competitive versus NADP, with K-i of 1 m u M, and by zinc ion, competitive versus glucose-6-phosphate, with K-i of 2 mu M. Crude enzyme extract catalyzed an appreciable rate of redu ction of NADP in the absence of added substrate, a ''nothing dehydroge nase'' activity. This activity was shown to be due to the presence of glucose-6-phosphate in the crude extract. It was calculated that cells of C. neoformans contain about 25 mu mol of glucose-6-phosphate per g ram, wet weight. (C) 1994 Academic Press, Inc.