The identification of a lysine residue reactive to pyridoxal-5-phosphate in the glycerol dehydrogenase from the thermophile Bacillus stearothermophilus.
The glycerol dehydrogenase (GDH) from Bacillus stearothermophilus is inactivated by incubation with pyridoxal-5-phosphate (PALP). The complex formed between the two can be trapped by reduction with sodium borohydride to yield a protein with an absorbance band at 325 nm and a fluorescence emission band at 430 nm, typical of trapped pyridoxal-5-phosphate moieties. Total loss of catalytic activity of the enzyme is associated with the modification of approximately one equivalent of the reagent; the incorporation of the reagent and the loss of activity can be prevented by the additional presence of the oxidised or reduced coenzyme. Peptides derived from the labelled protein have been sequenced and have identified Lys-97 as the reactive residue. Site-directed mutagenesis had been used to replace Lys-97 by a His residue. This mutated enzyme has no catalytic activity and fluorescence spectroscopy studies suggest that it is unable to bind NADH.
Department of Biochemistry, School of Biological Sciences, University of Southampton, UK.
L J
LJ
Paine
N
N
Perry
A G
AG
Popplewell
M G
MG
Gore
T
T
Atkinson
D000595:Amino Acid Sequence; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry; D010446:Peptide Fragments / Q000302:isolation & purification; D011732:Pyridoxal Phosphate / Q000737:chemistry* / Q000494:pharmacology; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry* / Q000235:genetics; D014357:Trypsin
D010446:Peptide Fragments; D009243:NAD; D011732:Pyridoxal Phosphate; D013401:Sugar Alcohol Dehydrogenases; C020672:glycerol dehydrogenase; D014357:Trypsin; D008239:Lysine