Active-site modification of glycerol-3-phosphate dehydrogenase by pyridoxal-5′-phosphate

Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) from rabbit skeletal muscle is inhibited by pyridoxal-5′-phosphate. The inhibition observed in steady-state kinetic studies is competitive with respect to dihydroxyacetone phosphate and uncompetitive with respect to NADH. Similar inhibition was found for a series of related compounds which in order of increasing effectiveness of inhibition were: 4-deoxypyridoxine < pyridoxal < pyridoxic acid < pyridoxal-5′-phosphate < pyridoxine and pyridoxamine-5′-phosphate. Pyridoxal-5′-phosphate also reacts slowly with the enzyme to produce an adduct which upon treatment with sodium borohydride results in irreversible modification of the enzyme. The nature of the adduct was investigated by titration of the enzyme with pyridoxal-5′-phosphate, uv-visible and fluorescence spectroscopy, amino acid analysis, and peptide mapping. All such studies are consistent with a single, highly reactive lysyl residue on each enzyme subunit. Protection of the lysyl residue against modification was afforded by the presence of NADH. The modified enzyme, on the other hand, possessed kinetic properties similar to the native enzyme including a nearly identical inhibition constant for pyridoxal-5′-phosphate. Pyridoxal-5′-phosphate, therefore, seems to have two sites of interaction on the enzyme: a reversible binding site competitive with substrate and a Schiff-base site protected by NADH. These properties of glycerol-3-phosphate dehydrogenase set it apart from functionally similar enzymes.