Characterisation of the Glycerol dehydrogenase from Bacillus stearothermophilus

Glycerol dehydrogenase (GDH) from Bacillus stearothermophilus is a NAD+/NADH dependent glycerol 2:oxidoreductase and therefore has the classification EC.1.1.1.6. This GDH has been shown to be a tetramer of identical subunits of Mr42kDa, requiring the presence of a single bound Zn2+ ion per subunit for activity and is A-stereospecific with respect to hydride transfer to and from the nicotinamide ring. In these respects GDH appears to belong to the family of alcohol/polyol metallo-dehydrogenases and it is proposed that GDH from B.stearothermophilus may be representative of a third distinct group within this classification. Removal of the metal ion from GDH results in the complete loss of activity which can then be regenerated by addition of several divalent metal ions eg. Zn2+, Co^2+, Cd^2+, Mn^2+ but not by Cu^2+ or Ni^2+. Co-factors were found to bind to the metal-depleted GDH which still has one NADH binding site per subunit with only a small reduction in affinity compared to that ppesent in the Zn^2+-metallo-GDH. However, the enhancement of the fluorescence at 430nm from NADH when bound to metallo-GDH of 25 (compared to the fluorescence of NADH in buffer) is more than that measured for the same complex formed with apoGDH (14 at pH7.4). This difference in fluorescence enhancement of NADH bound to apoGDH allowed the monitoring of Zn^2+ binding to this species. The process of Zn^2+ binding was determined to be second order with an on-rate of 1.4x10^3 M^-1s^-1 in the presence of enzyme-bound NADH and a predicted rate of 38x10^3M^-1s^-1 in the absence of NADH at pH6.0. A conformational change occurring to GDH subsequent to the removal of the bound metal was identified. This resulted in the loss of ability of metal-depleted GDH to rapidly regain activity on exposure to Zn^2+ ions, full reactivation requiring time periods in the region of 30-40 min. It is proposed that an equilibrium exists between two conformers of apoGDH, apoE^ which is able to bind metal andapoE which is not able to bind metal. This conformational equilibrium was found to be temperature and pH dependent,high temperatures and low pH favouring the apoE^ form. Chemical inactivation studies on apoGDH have implicated two histidinyl residues acting as ligands to the metal ion.The sole cysteine residue in GDH was found to be essential, its modification in apo-GDH resulting in the loss in the ability to regenerate activity from apoGDH on exposure to Zn^2+ ions. This loss of Zn^2+ reactivation was paralleled by tetramer dissociation. Therefore the cysteine residue could be acting as a Zn^2+ ligand or in stabilisation of the apoGDH tetramer. The sequence surrounding the cysteine residue was identified by carboxymethylation with iodo-[2-^3H]acetic acid, subsequent protein sequencing revealed the sequence TMAGGIPTIAAEAIAEKCEQTLFK. No homology to other published protein sequences have been detected to assist in the identification of the role of this cysteine residue. Similarly the N-terminal sequence of this GDH revealed no homologies to other published sequences. A map of the sequence to locate Met, Cys and Trp was constructed and this will be of assistance in more extensive sequencing studies.(DX86080)