Structural studies on glycerol dehydrogenase from Bacillus stearothermophilus

Glycerol dehydrogenase (GDH) (E.C.1.1.1.6.) from Bacillus stearothermophilus catalyses the reversible NAD⁺-dependent oxidation of glycerol to form dihydroxyacetone. The enzyme consists of four identical subunits each of M_r42000.

The binding of Ni²⁺, Cd²⁺ and Mn²⁺ to ^ApoGDH has been studied. The results demonstrate, that the dissociation constant for Cd²⁺, Ni²⁺ and Mn²⁺ are about 10, 20000 and 2000 times higher than that for Zn²⁺. The stability of the different metal derivatives has been determined by protein unfolding studies. These indicate that binding of Zn²⁺ leads to an increase in stabilisation of 7.0 kJ/mol in comparison to ^ApoGDH. The other metal enzyme complexes show also a considerable decrease in the conformational stability.

In order to identify histidine residue involved in metal ligation, three histidine mutants H271Q, H274Q and H291Q were produced and characterised.

The K_drates of Zn²⁺ ions from binary complexes made, using all histidine mutants produced, were determined. The data showed that the K_d rates for H274Q and the previously produced mutant H256Q are considerably increased.

This indicates that histidine residues at position 256 and 274 are probably involved in metal binding.

The protein folding pathway of the enzyme was studied. The refolding and reassociation was also studied in the presence of the chaperone GroEL. In these studies it was observed that the extent of reassociation is considerably increased in the presence of GroEL alone. The addition of ATP does not affect the extent of reassociation, but ATP increases the rate of reassociation slightly.

A molten globule state of GDH could be trapped under certain conditions. At pH 11.0 GDH showed a reduced tryptophan fluorescence and a higher quantum yield in ANS fluorescence.

University of Southampton

Krauss, Oliver

1996

Krauss, Oliver

Krauss, Oliver (1996) Structural studies on glycerol dehydrogenase from Bacillus stearothermophilus. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

In order to identify histidine residue involved in metal ligation, three histidine mutants H271Q, H274Q and H291Q were produced and characterised.

This indicates that histidine residues at position 256 and 274 are probably involved in metal binding.

A molten globule state of GDH could be trapped under certain conditions. At pH 11.0 GDH showed a reduced tryptophan fluorescence and a higher quantum yield in ANS fluorescence.

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