Chemical modification, mutagenesis and characterisation of the glycerol dehydrogenase from Bacillus stearothermophilus
Chemical modification, mutagenesis and characterisation of the glycerol dehydrogenase from Bacillus stearothermophilus
The glycerol dehydrogenase (E.C. 1.1.1.6) (GDH) from Bacillus stearothermophilus is a tetramer of identical subunits (Mr im 42,000) requiring one zinc ion per subunit for catalytic activity. Loss of the metal ion permits the enzyme to undergo a conformational change at low temperatures which can result in dissociation of the subunits. A study by selective chemical modification of amino acid residues which have a structural, binding or catalytic role in the enzyme has revealed the presence of two lysine residues reactive to pyridoxal 5'-phosphate. Kinetic analysis of the modification process revealed that the reaction proceeds through a two step mechanism. An initial non-covalent complex is rapidly formed which is followed by a slower formation of a Schiff's base which results in inactivation of the enzyme. Labelling experiments followed by tryptic digestion, peptide isolation and sequencing has led to the identificaiton of one of these residues as lysine 97. The modification of this residue can be protected by the additional presence of NAD+ or NADH in the reaction mixture. The use of other reagents such as Phenylglyoxal and Woodwards reagent K has led to the conclusion that there are one or more arginine residues and one or more aspartate/glutamate residues respectively located in or close to the coenzyme binding site of GDH. Site-directed mutagenesis of the reactive lysine residue 97 to a histidine residue produced an inactive mutant enzyme. However, mutagenesis to an arginine residue produced an enzyme capable of supporting catalytic activity but of a much lower substrate and cofactor affinity. This enzyme was expressed mainly as a monomer, was able to bind zinc ions and was activated by incubation at temperatures above 35oC. Furthermore, K97R GDH was shown to undergo a reversible conformational change in response to changes in pH and temperature, similar to that seen in native apoGDH. However, K97R GDH undergoes this change when the metal ion is bound to the enzyme. Enzymic activity of K97R GDH could also be increased by incubation of the enzyme with NAD+ , prior to assay. Substitution of lysine 97 by an arginine residue appears to compromise the tetrameric integrity of the enzyme. This may result from a direct perturbation of subunit conformation. Mutation of cysteine 208 to an alanine residue results in the enzyme being isolated as monomers which are catalytically inactive suggesting that this residue also plays an important role in formation and stabilisation of the oligomer.
University of Southampton
1992
Paine, Lisa Jane
(1992)
Chemical modification, mutagenesis and characterisation of the glycerol dehydrogenase from Bacillus stearothermophilus.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The glycerol dehydrogenase (E.C. 1.1.1.6) (GDH) from Bacillus stearothermophilus is a tetramer of identical subunits (Mr im 42,000) requiring one zinc ion per subunit for catalytic activity. Loss of the metal ion permits the enzyme to undergo a conformational change at low temperatures which can result in dissociation of the subunits. A study by selective chemical modification of amino acid residues which have a structural, binding or catalytic role in the enzyme has revealed the presence of two lysine residues reactive to pyridoxal 5'-phosphate. Kinetic analysis of the modification process revealed that the reaction proceeds through a two step mechanism. An initial non-covalent complex is rapidly formed which is followed by a slower formation of a Schiff's base which results in inactivation of the enzyme. Labelling experiments followed by tryptic digestion, peptide isolation and sequencing has led to the identificaiton of one of these residues as lysine 97. The modification of this residue can be protected by the additional presence of NAD+ or NADH in the reaction mixture. The use of other reagents such as Phenylglyoxal and Woodwards reagent K has led to the conclusion that there are one or more arginine residues and one or more aspartate/glutamate residues respectively located in or close to the coenzyme binding site of GDH. Site-directed mutagenesis of the reactive lysine residue 97 to a histidine residue produced an inactive mutant enzyme. However, mutagenesis to an arginine residue produced an enzyme capable of supporting catalytic activity but of a much lower substrate and cofactor affinity. This enzyme was expressed mainly as a monomer, was able to bind zinc ions and was activated by incubation at temperatures above 35oC. Furthermore, K97R GDH was shown to undergo a reversible conformational change in response to changes in pH and temperature, similar to that seen in native apoGDH. However, K97R GDH undergoes this change when the metal ion is bound to the enzyme. Enzymic activity of K97R GDH could also be increased by incubation of the enzyme with NAD+ , prior to assay. Substitution of lysine 97 by an arginine residue appears to compromise the tetrameric integrity of the enzyme. This may result from a direct perturbation of subunit conformation. Mutation of cysteine 208 to an alanine residue results in the enzyme being isolated as monomers which are catalytically inactive suggesting that this residue also plays an important role in formation and stabilisation of the oligomer.
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Published date: 1992
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Local EPrints ID: 461733
URI: http://eprints.soton.ac.uk/id/eprint/461733
PURE UUID: ac428300-e68d-460c-a2d3-9736745ec156
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Date deposited: 04 Jul 2022 18:53
Last modified: 04 Jul 2022 18:53
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Author:
Lisa Jane Paine
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