Active-site histidines in recombinant cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase examined by site-directed mutagenesis
✍ Scribed by Robert L. Haining; Bruce A. McFadden
- Publisher
- Springer
- Year
- 1994
- Tongue
- English
- Weight
- 903 KB
- Volume
- 41
- Category
- Article
- ISSN
- 0166-8595
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✦ Synopsis
The functions of H i s 291 , H i s 295 and H i s 324 at the active-site of recombinant A. nidulans ribulose-1,5-bisphosphate carboxylase/oxygenase have been explored by site-directed mutagenesis. Replacement of His 291 by K or R resulted in unassembled proteins, while its replacement by E, Q or N resulted in assembled but inactive proteins. These results are in accord with a metal ion-binding role of this residue in the activated ternary complex by analogy to x-ray crystallographic analyses of tobacco and spinach enzymes.
H i s 324 (H327 in spinach), which is located within bonding distance of the 5-phosphate of bound bi-substrate analog 2-carboxyarabinitol 1,5-bisphosphate in the crystal structures, has been substituted by A, K, R, Q and N. Again with the exception of the H324K and R variants, these changes resulted in detectable assembled protein.
The mutant H324A protein exhibited no detectable carboxylase activity, whereas the H324Q and H324N changes resulted in purifiable holoenzyme with 2.0 and 0.1% of the recombinant wild-type specific carboxylase activity, respectively. These results are consistent with a phosphate binding role for this residue.
The replacement of His 295, which has been suggested to aid in phosphate binding, with Ala in the A. nidulans enzyme leads to a mutant with 5.8% of the recombinant wild-type carboxylase activity. All other mutations at this position resulted in unassembled proteins. Purified H295A and H324Q enzymes had elevated Km(RuBP) values and unchanged CO2/O 2 specificity factors compared to recombinant wild-type.