Reiersen H, Lindstad R I, McKinley-McKee J S
Biochemical Institute, University of Oslo, Norway.
Arch Biochem Biophys. 1994 Jun;311(2):450-6. doi: 10.1006/abbi.1994.1261.
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.
焦磷酸及其他几种金属螯合剂可使绵羊肝脏山梨醇脱氢酶失活。焦磷酸、三聚磷酸和一些双膦酸盐通过涉及形成可逆复合物的饱和动力学使该酶失活。本文提出了焦磷酸介导山梨醇脱氢酶失活的机制。稳态动力学表明，焦磷酸不与山梨醇竞争结合催化锌原子，也不与NAD竞争结合阴离子结合位点。E-NAD-焦磷酸（PPi）复合物的形成以及NADH对失活的非竞争性保护支持了后者。焦磷酸使酶失活的速率随pH值降低而增加。失活对pH的依赖性表明，游离酶和酶-PPi复合物中一个pKa为6.9的基团参与其中。由于几种与锌结合的可逆抑制剂不能防止焦磷酸失活，因此所获得的pKa值被认为不是指与锌结合的水分子的电离，而是初步认为是活性位点组氨酸残基的pKa值。Zn2+离子的保护和再激活表明酶失活是由于催化锌原子的丢失。
