Chemical modification of specific active site amino acid residues of Enterobacter aerogenes glycerol dehydrogenase.
Enterobacter aerogenes glycerol dehydrogenase (G1DH EC 1.1.1.6), a tetrameric NAD+ specific enzyme catalysing the interconversion of glycerol and dihydroxyacetone, was inactivated on reaction with pyridoxal 5-phosphate (PLP) and o-phthalaldehyde (OPA). Fluorescence spectra of PLP-modified, sodium borohydride-reduced G1DH indicated the specific modification of epsilon-amino groups of lysine residues. The extent of inhibition was concentration and time dependent. NAD+ and NADH provided complete protection against enzyme inactivation by PLP, indicating the reactive lysine is at or near the coenzyme binding site. Modification of G1DH by the bifunctional reagent OPA, which reacts specifically with proximal epsilon-NH2 group of lysines and -SH group of cysteines to form thioisoindole derivatives, inactivated the enzyme. Molecular weight determinations of the modified enzyme indicated the formation of intramolecular thioisoindole formation. Glycerol partially protected the enzyme against OPA inactivation, whereas NAD+ was ineffective. These results show that the lysine involved in the OPA reaction is different from the PLP-reactive lysine, which is at or near the coenzyme binding site. DTNB titration showed the presence of only a single cysteine residue per monomer of G1DH. This could be participating with a proximal lysine residue to form a thioisoindole derivative observed as a result of OPA modification.
D000596:Amino Acids / Q000032:analysis / Q000737:chemistry*; D001665:Binding Sites; D021902:Enterobacter aerogenes / Q000201:enzymology*; D004791:Enzyme Inhibitors; D005990:Glycerol / Q000494:pharmacology; D009243:NAD / Q000494:pharmacology; D011732:Pyridoxal Phosphate; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry*; D009764:o-Phthalaldehyde
D000596:Amino Acids; D004791:Enzyme Inhibitors; D009243:NAD; D011732:Pyridoxal Phosphate; D009764:o-Phthalaldehyde; D013401:Sugar Alcohol Dehydrogenases; C020672:glycerol dehydrogenase; D005990:Glycerol