Rapid purification and biochemical characterization of glucose kinase fromStreptomyces peucetius var. caesius

Glucose kinase catalyzes the ATP-dependent phosphorylation of glucose. Stre ptomyces peucetius var. caesius glucose kinase was purified 292-fold to hom ogeneity. The enzyme has cytosolic localization and is composed of four ide ntical subunits, each of 31 kDa. The purified enzyme easily dissociates int o dimers. However, in the presence of 100 mM glucose the enzyme maintains i ts tetrameric form. Maximum activity was found at 42 degreesC and pH 7.5. I soelectric focusing of the enzyme showed a pl of 8.4. The N- and C-terminal amino acid sequences were MGLTIGVD and VY-FAREPDPIM, respectively. The kin etic mechanism of S. peucetius var. caesius glucose kinase appears to be a rapid equilibrium ordered type, i.e., ordered addition of substrates to the enzyme, where the first substrate is D-glucose. The K-m values for D-gluco se and MgATP(2-) were 1.6 +/- 0.2 and 0.8 +/- 0.1 mM, respectively. Mg2+ in excess of 10 mM inhibits enzyme activity. (C) 2001 Academic Press.