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 a re, however, conflicting views as to whether the lysine residues are involv ed in Schiff's base formation with catalytic intermediates, stabilization o f negatively charged groups or the carbonyl group of 2-oxoglutarate during catalysis, or some other function. We have expanded on these speculations b y constructing a series of cassette mutations at Lys130, a residue that has been speculated to be responsible for the activity of GDH and the inactiva tion of GDH by pyridoxal 5'-phosphate (PLP). For these studies, a 1557-bp g ene that encodes human GDH has been synthesized and inserted into Escherich ia coli expression vectors. The mutant enzymes containing Glu, Gly, Met, Se r, or Tyr at position 130, as well as the wild-type human GDH encoded by th e synthetic gene, were efficiently expressed as a soluble protein and are i ndistinguishable from that isolated from human and bovine tissues. Despite an approximately 400-fold decrease in the respective apparent V-max of the Lys130 mutant enzymes, apparent K-m 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 L ys130 plays an important role in PLP binding. The results with analogs of P LP suggest that the aldehyde moiety of PLP, but not the phosphate moiety, i s required for efficient binding to GDH.