, Volume 183, Issue 4, pp 597–603 | Cite as

Proteolysis and transition-state-analogue binding of mutant forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii

  • Zhixiang Chen
  • Robert J. Spreitzer


Trypsin digestion reduces the sizes of both the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC from the green alga Chlamydomonas reinhardtii. Incubation of either CO2/Mg2+ -activated or nonactivated enzyme with the transition-state analogue carboxyarabinitol bisphosphate protects a trypsin-sensitive site of the large subunit, but not of the small subunit. Incubation of the nonactivated enzyme with ribulosebisphosphate (RuBP) provided the same degree of protection. Thus, the very tight binding that is a characteristic of the transitionstate analogue is apparently not required for the protection of the trypsin-sensitive site of the large subunit. Mutant enzymes that have reduced CO2/O2 specificities failed to bind carboxyarabinitol bisphosphate tightly. However, their large-subunit trypsin-sensitive sites could still be protected. The Km values for RuBP were not significantly changed for the mutant enzymes, but the Vmax values for carboxylation were reduced substantially. These results indicate that the failure of the mutant enzymes to bind the transition-state analogue tightly is primarily the consequence of an impairment in the second irreversible binding step. Thus, in all of the mutant enzymes, defects appear to exist in stabilizing the transition state of the carboxylation step, which is precisely the step proposed to influence the CO2/O2 specificity of Rubisco.

Key words

Chlamydomonas Chloroplast mutants Photosynthesis (Rubisco) Ribulose-1,5-bisphosphate carboxylase/oxygenase 

Abbreviations and Symbols


2-carboxyarabinitol 1,5-bisphosphate


2,3-enediolate of ribulose 1,5-bisphosphate


Km for CO2


Km for O2


ribulose-1,5-bisphosphate carboxylase/oxygenase


ribulose 1,5-bisphosphate


Vmax for carboxylation


Vmax for oxygenation


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Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Zhixiang Chen
    • 1
  • Robert J. Spreitzer
    • 1
  1. 1.Department of BiochemistryUniversity of NebraskaLincolnUSA

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