The inactivation of sheep liver sorbitol dehydrogenase by pyrophosphate and some analogous metal chelators.
Pyrophosphate and several other metal chelators are shown to inactivate sheep liver sorbitol dehydrogenase. Pyrophosphate, tripolyphosphate, and some bisphosphonates inactivate the enzyme by saturation kinetics involving the formation of a reversible complex. A mechanism for the pyrophosphate-mediated inactivation of sorbitol dehydrogenase is proposed. Steady-state kinetics show that pyrophosphate does not compete with sorbitol for binding to the catalytic zinc atom or with NAD for binding to the anion binding site. The latter is supported by the formation of an E-NAD-pyrophosphate (PPi) complex and by the noncompetitive protection of NADH against inactivation. The rate of enzyme inactivation by pyrophosphate increases with decreasing pH. The pH dependence of the inactivation indicates that a group with a pKa of 6.9 in the free enzyme and in the enzyme-PPi complex is involved. As several zinc-binding reversible inhibitors do not afford protection against pyrophosphate inactivation, the pKa values obtained are considered not to refer to the ionization of the zinc-bound water molecule, but are tentatively suggested to be those of an active site histidine residue. Protection and reactivation by Zn2+ ions indicate that enzyme inactivation results from the loss of the catalytic zinc atom.
Reiersen
H
H
Biochemical Institute, University of Oslo, Norway.
Lindstad
R I
RI
McKinley-McKee
J S
JS
D000818:Animals; D002614:Chelating Agents / Q000494:pharmacology*; D011756:Diphosphates / Q000494:pharmacology*; D004789:Enzyme Activation; D007700:Kinetics; D007064:L-Iditol 2-Dehydrogenase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D015394:Molecular Structure; D011122:Polyphosphates / Q000494:pharmacology; D012756:Sheep; D013329:Structure-Activity Relationship; D015032:Zinc / Q000494:pharmacology
D002614:Chelating Agents; D011756:Diphosphates; D011122:Polyphosphates; D007064:L-Iditol 2-Dehydrogenase; D015032:Zinc