J. Biol. Chem., Vol. 261, Issue 23, 10659-10666, 08, 1986
Reaction of the 2',3'-dialdehyde derivative of NADPH at a nucleotide site of bovine liver glutamate dehydrogenase
RH Lark and RF Colman
The 2',3'-dialdehyde derivative of NADPH (oNADPH) acts as a coenzyme for
the reaction catalyzed by bovine liver glutamate dehydrogenase. Incubation
of 250 microM oNADPH with enzyme for 300 min at 30 degrees C and pH 8.0
yields covalent incorporation of 1.0 mol of oNADPH/mol of enzyme subunit.
The modified enzyme has a functional catalytic site and is activated by
ADP, but is no longer inhibited by high NADH concentrations and exhibits
decreased sensitivity to GTP inhibition. Using the change in inhibition by
600 microM NADH or 1 microM GTP to monitor the reaction leads to rate
constants of 44.0 and 41.5 min-1 M- 1, respectively, suggesting that loss
of inhibition by the two regulatory compounds results from reaction by
oNADPH at a single location. The oNADPH incorporation is proportional to
the decreased inhibition by 600 microM NADH or 1 microM GTP, extrapolating
to less than 1 mol of oNADPH/mol of subunit when the maximum change in NADH
or GTP inhibition has occurred. Modified enzyme is still 93% inhibited at
saturating levels of GTP, although its K1 is increased 20-fold to 4.6
microM. The kinetic effects caused by oNADPH are not prevented by alpha-
ketoglutarate, ADP, 5 mM NADH, or 200 microM GTP alone, but are prevented
by 5 mM NADH with 200 microM GTP. Incorporation of oNADPH into enzyme at
255 min is 0.94 mol/mol of peptide chain in the absence of ligands but only
0.53 mol/mol of peptide chain in the presence of the protectants 5 mM NADH
plus 200 microM GTP. These results indicate that oNADPH modifies
specifically about 0.4-0.5 sites/enzyme subunit or about 3 sites/enzyme
hexamer and that reaction occurs at a GTP- dependent inhibitory NADH site
of glutamate dehydrogenase.
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