Putative functional role for the invariant aspartate 263 residue of Rhodospirillum rubrum Rubisco.
Although aspartate residue D263 of Rhodospirillum rubrum Rubisco is close to the active site and invariant in all reported Rubiscos, its possible functional and structural roles in Rubisco activity have not been investigated. We have mutagenised D263 to several selected amino acids (asparagine, alanine, serine, glutamate, and glutamine) to probe possible roles in facilitating proton movements within the active site and maintaining structural positioning of key active-site groups. The mutants have been characterized by kinetic methods and by differential scanning calorimetry (DSC) to examine the effects of the substitutions on the stability of the folded state. We show that D263 is essential for maintaining effective levels of catalysis with the mutations reducing carboxylation variously by up to 100-fold but having less than 10% effect on the carboxylase/oxygenase specificity of the catalytic reaction. Removing the charge of the residue 263 side chain significantly strengthens binding of the activating (carbamylating) CO(2) molecule. In contrast, a charge on the 263 site has only a small influence on binding of the positively charged Mg(2+) ion, suggesting that the local protein structure provides different shielding of the formal charges on the Mg(2+) ion and the epsilon-lysine group of K191. Interestingly, introduction of an internal cavity (D263S and D263A) and insertion of an extra -CH(2)- group (D263E and D263Q) have opposite effects on catalysis, the former relatively small and the latter much larger, suggesting that the extra side-chain group induces a specific structural distortion that inhibits formation of the transition state. As the DSC results show that the mutations only slightly increase the kinetic stability of the folded state, we conclude that the rate-limiting (activated) step of unfolding involves substantial unfolding of the structure but not in the region of site 263. In summary, interaction of D263 with H287 of a largely electrostatic nature appears critical for maintaining correct positioning of catalytic groups in the active site. The conservation of D263 can thus be accounted for by its contribution to the maintenance of a finely tuned structure in this region abutting the active site.
Liggins
John R
JR
Molecular Plant Physiology Group, Research School of Biological Sciences, John Curtin School of Medical Research, Australian National University, Canberra ACT 0200, Australia.
Gready
Jill E
JE
D019943:Amino Acid Substitution / Q000502:physiology; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002152:Calorimetry, Differential Scanning; D002245:Carbon Dioxide / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011489:Protein Denaturation / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology* / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012274:Ribulosephosphates / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D013816:Thermodynamics
D011994:Recombinant Proteins; D012274:Ribulosephosphates; D002245:Carbon Dioxide; C001933:ribulose-1,5 diphosphate; D001224:Aspartic Acid; D012273:Ribulose-Bisphosphate Carboxylase; D008274:Magnesium