Purification and properties of crystalline 3-hydroxybutyrate dehydrogenase from Rhodopseudomonas spheroides

Abstract

The purification and crystallization of 3-hydroxybutyrate dehydrogenase from extracts of R. spheroides is described. The molecular weight was calculated to Be 85000 by sedimentation equilibrium. Although the enzyme is stable at 0-4[degree], dilute solutions are rapidly inactivated at 37[degree]; NADH2 or Ca2+ ions prevent this inactivation. The enzyme is extremely sensitive to mercurials, but can be protected by NADH2 or Ca2+ ions. From studies on p-hydroxymercuribenzoate binding it is estimated that the enzyme contains 5-6 moles of rapidly reacting thiol groups/mole. D-Lactate and DL-2-hydroxybutyrate are competitive inhibitors of D-3-hydroxy-butyrate oxidation. The properties of the crystalline enzyme are compared with those of 3-hydroxybutyrate dehydrogenase preparations from other sources.