Inactivation of human glutamate dehydrogenase by aluminum.
Aluminum inactivated glutamate dehydrogenase (GDH) by a pseudo-first-order reaction at micromolar concentrations. A double-reciprocal plot gave a straight line with a k(inact) of 2.7 min(-1) and indicated the presence of a binding step prior to inactivation. The inactivation was strictly pH dependent and a marked increase in sensitivity to aluminum was observed as the pH decreased. At a pH higher than 8.5, no inactivation was observed. The completely inactivated GDH contained 2 mol of aluminum per mole of enzyme subunit monomer. When preincubated with enzyme, several chelators such as citrate, NaF, N-(2-hydroxyethyl) ethylenediaminetriacetic acid or ethylenediaminetriacetic acid efficiently protected the enzyme against the aluminum inactivation. In a related experiment, only citrate and NaF released the aluminum from the completely inactivated aluminum-enzyme complex and fully recovered the enzyme activity. Ferritin, NADP+, or nerve growth factor did not show any effects on the recovery of the aluminum-inactivated GDH activity. The dissociation constant for the aluminum-enzyme complex was calculated to be 5.3 microM. Although aluminum has been known to form a complex with nucleotides, no such effects were observed in the inactivation of GDH by aluminum as determined using GDHs mutated at the ADP-binding site, NAD+-binding site or GTP-binding site. Circular dichroism studies showed that the binding of aluminum to the enzyme induced a decrease in alpha helices and beta sheets and an increase in random coil. Therefore, inactivation of GDH by aluminum is suggested to be due to the conformational change induced by aluminum binding. These results suggest a possibility that aluminum-induced alterations in enzymes of the glutamate system may be one of the causes of aluminum-induced neurotoxicity.
Yang
S-J
SJ
Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, 388-1 Poongnap-dong, Songpa-ku, Seoul 138-736, Korea.
Huh
J-W
JW
Lee
J E
JE
Choi
S Y
SY
Kim
T U
TU
Cho
S-W
SW
D000535:Aluminum / Q000378:metabolism / Q000633:toxicity*; D002614:Chelating Agents / Q000494:pharmacology; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000187:drug effects; D005969:Glutamate Dehydrogenase / Q000187:drug effects* / Q000378:metabolism; D018698:Glutamic Acid / Q000633:toxicity; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009696:Nucleic Acids / Q000378:metabolism
D002614:Chelating Agents; D009696:Nucleic Acids; D018698:Glutamic Acid; D000535:Aluminum; D005969:Glutamate Dehydrogenase