Comparison of properties of purified human and rabbit serum paraoxonases.

Abstract

Rabbits have several times greater serum paraoxonase activity than humans, or other mammalian species. We have studied the basis for this difference. Studies of substrate specificity, disulfide bonds, sugar chains, and phospholipid stimulation show similar properties between two species. Conversely, there is some evidence that the rabbit enzyme is more stable than the human enzyme, including heat inactivation, zymogram separation (on SDS-PAGE gel), and EDTA or Chelex 100 treatment. Although these two paraoxonases have considerable structural homology with 85% identical amino acid residues, there is a significant difference in that the rabbit esterase binds Ca$\sp{2+}$ more tightly than the human esterase. Ca$\sp{2+}$ is required for catalytic activity and also stabilizes the enzymes in their active forms. Other metals such as Zn$\sp{2+}$, or Cd$\sp{2+}$ can substitute for Ca$\sp{2+}$ maintaining both enzymes in their active forms, but these other metals do not confer catalytic activity. Recently, our studies have focused on details of the calcium binding. Equilibrium dialysis studies have shown that both human and rabbit enzymes possess two Ca$\sp{2+}$-binding sites. Determination of both independent-site and stepwise-equilibrium constants show that, in human paraoxonase, the dissociation constant (Kd$\sb1$) for the higher affinity site is $6.7\pm0.5\times10\sp{-7}$ M, and Kd$\sb2$ for the lower affinity one is $1.22\pm0.4\times10\sp{-4}$ M; whereas, for rabbit paraoxonase, Kd$\sb1$ is $4.5\pm0.7\times10\sp{-8}$ M, and Kd$\sb2$ it is $1.9\pm1.0\times10\sp{-5}$ M. The Hill coefficient for the human enzyme is 0.7, and for the rabbit enzyme is 0.4. We were also able to show that the higher affinity Ca$\sp{2+}$-binding site, which is irreversible, is essential for the stability of the enzyme, whereas the lower affinity one, which is reversible, is involved in the catalytic activity of the enzyme. We conclude that the active centers of the two paraoxonases are very similar, but rabbit paraoxonase binds calcium more tightly, is a more stable enzyme, and is maintained at a higher steady-state concentration in serum than human paraoxonase.