Purification and properties of glucose 6-phosphate dehydrogenase from Aspergillus aculeatus.
Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) was purified from Aspergillus aculeatus, a filamentous fungus previously isolated from infected tongue of a patient. The enzyme, apparently homogeneous, had a specific activity of 220 units mg(-1), a molecular weight of 105,000 +/- 5,000 Dal by gel filtration and subunit size of 52,000 +/- 1,100 Dal by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The substrate specificity was extremely strict, with glucose 6-phosphate (G6P) being oxidized by nicotinamide adenine dinucleotide phosphate (NADP) only. At assay pH of 7.5, the enzyme had K(m) values of 6 microM and 75 microM for NADP and G6P respectively. The k(cat) was 83 s(-1). Steady-state kinetics at pH 7.5 produced converging linear Lineweaver-Burk plots as expected for ternary-complex mechanism. The patterns of product and dead-end inhibition suggested that the enzyme can bind NADP and G6P separately to form a binary complex, indicating a random-order mechanism. The enzyme was irreversibly inactivated by heat in a linear fashion, with G6P providing a degree of protection. Phosphoenolpyruvate (PEP), adenosinetriphosphate (ATP), and fructose 6-phosphate (F6P), in decreasing order, are effective inhibitors. Zinc and Cobalt ions were effective inhibitors although cobalt ion was more potent; the two divalent metals were competitive inhibitors with respect to G6P, with Ki values of 6.6 microM and 4.7 microM respectively. It is proposed that inhibition by divalent metal ions, at low NADPH /NADP ratio, is another means of controlling pentosephosphate pathway.
Ibraheem
Omodele
O
Department of Biochemistry, Obafemi Awolowo University, Ile-Ife, Nigeria.
Adewale
Isaac Olusanjo
IO
Afolayan
Adeyinka
A
D001230:Aspergillus / Q000201:enzymology*; D002413:Cations, Divalent / Q000378:metabolism; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D005656:Fungal Proteins; D005954:Glucosephosphate Dehydrogenase