Is pyridoxal 5'-phosphate an affinity label for phosphate-binding sites in proteins?: The case of bovine glutamate dehydrogenase.
The effects of pyridoxal 5'-phosphate (PalP) on ox liver glutamate dehydrogenase (94% inactivation by 1.8 mM reagent at pH 7 and 25 degrees C) have been compared with those of three analogues, 5'-deoxypyridoxal (96% inactivation), pyridoxal 5'-sulphate (97%) and pyridoxal 5-methylsulphonate (94%), in order to establish whether PalP acts as an affinity label for this enzyme. Like PalP and unlike pyridoxal, which is a much less potent inactivator, none of the analogues has a free 5'-OH group to cyclize with the aldehyde function. The result with 5'-deoxypyridoxal shows that a negative charge, such as that of the phosphate group, is not required for efficient inactivation. With all four reagents, addition of an excess of cysteine or lysine led to 90-100% re-activation over 3-20 h. Dialysis also caused reactivation to a similar extent. A combination of 2.15 mM NADH, 1 mM GTP and 10 mM 2-oxoglutarate gave complete protection against PalP, but only partial protection against the analogues. 5'-Deoxypyridoxal still caused 20-25% inactivation in the presence of the protection mixture. Absorbance measurements after reduction with NaBH4 show the characteristic features of a reduced Schiff's base and allowed estimation of the extent of reaction. With all the reagents the protection mixture decreased incorporation by about 1 mol/mol, but levels of incorporation without protection varied from about 2 mol/mol for PalP up to about 5 mol/mol for 5'-deoxypyridoxal. The labelling at additional sites may explain the residual inactivation in the presence of potent protecting agents.
Valinger
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Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, U.K.
Engel
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PC
Metzler
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DE
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000737:chemistry*; D013329:Structure-Activity Relationship
D000345:Affinity Labels; D010710:Phosphates; D011732:Pyridoxal Phosphate; D005969:Glutamate Dehydrogenase