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Kurzfassung
The 45-3B mutant of Chlamydomonas reinhardtii has a mutation in the chloroplast large-subunit gene of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) that causes valine-331 to be replaced by alanine. This mutation causes a photosynthesis-deficient, acetate-requiring phenotype by reducing the specific activity of the Rubisco enzyme by 95% and the CO$\sb2$/O$\sb2$ specificity by 37%. Revertant selection identified two intragenic suppression mutations, named S40-9D and S61-2J, that cause threonine-342 to be replaced by isoleucine and glycine-344 to be replaced by serine, respectively. Both of these amino acid substitutions appear to complement the original 45-3B mutation through steric interaction to improve the specific activity and CO$\sb2$/O$\sb2$ specificity of the mutant enzyme.
Temperature-sensitive mutant 68-4PP resulted from a chloroplast mutation that causes a phenylalanine-for-leucine substitution at residue 290 in the Rubisco large subunit. The mutant Rubisco enzyme had about 50% of wild-type enzyme activity and its CO$\sb2$/O$\sb2$ specificity was reduced by 13%. Reversion experiments at the restrictive temperature (35$\sp\circ$C) identified a nuclear mutation which suppressed the temperature-sensitive phenotype of 68-4PP by enhancing both the specific activity and protein level of the mutant Rubisco enzyme. More significantly, the reduced CO$\sb2$/O$\sb2$ specificity factor of the 68-4PP mutant enzyme was restored to the wild-type level by this nuclear mutation. DNA sequencing showed that this mutation was not in either of the two Rubisco small-subunit genes, suggesting that some other nuclear-encoded protein is able to influence the structure and, in turn, the CO$\sb2$/O$\sb2$ specificity of Rubisco.
Binding assays with the transition-state analogue 2-carboxyarabinitol-1,5-bisphosphate, in combination with other biochemical analyses, revealed that the mutant forms of Rubisco with reduced CO$\sb2$/O$\sb2$ specificities had defects in stabilizing the transition state of the partial reaction of carboxylation. This observation was consistent with a prediction derived from transition state theory which indicates that the relative stabilities of the transition states for the competitive partial reactions of carboxylation and oxygenation determine the CO$\sb2$/O$\sb2$ specificity of Rubisco.
