Cassette mutagenesis of lysine 130 of human glutamate dehydrogenase. An essential residue in catalysis.
It has been suggested that reactive lysine residue(s) may play an important role in the catalytic activities of glutamate dehydrogenase (GDH). There are, however, conflicting views as to whether the lysine residues are involved in Schiff's base formation with catalytic intermediates, stabilization of negatively charged groups or the carbonyl group of 2-oxoglutarate during catalysis, or some other function. We have expanded on these speculations by constructing a series of cassette mutations at Lys130, a residue that has been speculated to be responsible for the activity of GDH and the inactivation of GDH by pyridoxal 5'-phosphate (PLP). For these studies, a 1557-bp gene that encodes human GDH has been synthesized and inserted into Escherichia coli expression vectors. The mutant enzymes containing Glu, Gly, Met, Ser, or Tyr at position 130, as well as the wild-type human GDH encoded by the synthetic gene, were efficiently expressed as a soluble protein and are indistinguishable from that isolated from human and bovine tissues. Despite an approximately 400-fold decrease in the respective apparent Vmax of the Lys130 mutant enzymes, apparent Km values for NADH and 2-oxoglutarate were almost unchanged, suggesting the direct involvement of Lys130 in catalysis rather than in the binding of coenzyme or substrate. Unlike the wild-type GDH, the mutant enzymes were unable to interact with PLP, indicating that Lys130 plays an important role in PLP binding. The results with analogs of PLP suggest that the aldehyde moiety of PLP, but not the phosphate moiety, is required for efficient binding to GDH.
Cho
S W
SW
Department of Biochemistry, University of Ulsan College of Medicine, Seoul, Korea. swcho@www.amc.seoul.kr
Yoon
H Y
HY
Ahn
J Y
JY
Lee
E Y
EY
Lee
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D011732:Pyridoxal Phosphate; D005969:Glutamate Dehydrogenase; D008239:Lysine