Initial-rate studies of a thermophilic glucokinase from Bacillus stearothermophilus

Abstract

The initial rates of phosphorylation of glucose catalysed by glucokinase from Bacillus stearothermophilus were measured over a wide range of glucose, MgATP2-, MgADP- and glucose 6-phosphate concentrations. The results of the effects of the inhibitors on the initial rates suggest that the reaction mechanism is essentially the ordered Bi Bi, in which glucose adds to the enzyme before MgATP2- and glucose 6-phosphate is released from the enzyme after the dissociation of MgADP-, and also suggest that the final step in which glucose 6-phosphate is released is irreversible. For many reaction schemes, the rate equations were derived on the basis of the pseudo-steady-state assumption and were used to correlate the experimental rate data. From this result, we concluded that the reaction obeys the ordered mechanism accompanied by the formation of a non-productive ternary complex, glucose-MgADP--enzyme. By using the experimental Dalziel coefficients phi i, some kinetic parameters were evaluated. The enzyme was characterized by the thermal stability and the low Michaelis constant, the values of which were 54 microM for glucose and 32 microM for MgATP2-.

Biochemistry and regulatory functions of bacterial glucose kinases

“…ATP-Glks, have molecular weights in a range of 24 to 33 kDa. Most optimal pH values are around 7.5, but in Bacillus stearothermophilus, we can observe an optimum pH value of 9.0 [24,15] and a pH value of 6.2 for the archaeon A. pernix [18]. km values for ATP ranged from 1.46 to 0.06 mM, and for glucose from 1.6 to 0.044 mM.…”

Thermostable Proteins as Probe for the Design of Advanced Fluorescence Biosensors

“…These enzymes are already in use as biocatalysts in industrial processes (Jaenicke et al 1996;Sthal 1993). A glucokinase from the thermophilic organism Bacillus stearothermophilus has been characterized and is known to display long term stability (Goward et al 1987;Ishikawa et al 1987;Tomita et al 1990). Hence we evaluated the use of this glucokinase (BSGK) in the absence of ATP as a non-consuming glucose sensor.…”

Glucose biosensors as models for the development of advanced protein-based biosensors

“…39 BSGK has a single cysteine residue located near the active site. 40 We labeled the residue with a sulfidryl-reactive fluorophore IA-ANS. The emission of the labeled protein was near 460 nm.…”