Quaternary structure and oxygenase activity of D-ribulose-1,5-bisphosphate carboxylase from Hydrogenomonas eutropha

Abstract

Electrophoretically homogeneous ribulose-1,5-bisphosphate (RuBP) carboxylase was obtained from autotrophically grown H. eutropha by sedimentation of the 105,000 .times. g supernatant in a discontinuous sucrose gradient and by (NH4)2SO4 fractionation followed by another sucrose gradient centrifugation. The MW of the enzyme determined by light scattering was 490,000 .+-. 15,000. The enzyme could be dissociated by sodium dodecyl sulfate into 3 types of subunits, and the MW (.+-. 10%) could be measured. There were 2 species of large subunits, L and L'' (MW 56,000 and 52,000, respectively) and 1 species of small subunits [S] (MW 15,000). The mole ratio of L to L'' was 5:3, and the overall mole ratio of the small to large subunits was 1.08. The simplest quaternary structure of the enzyme is L5L''3S8. The enzyme contained RuBP oxygenase activity as evidenced by the O2-dependent production of phosphoglycolate and 3-phosphoglyceric acid in equimolar quantities from RuBP.