Mutagenesis at two distinct phosphate-binding sites unravels their differential roles in regulation of Rubisco activation and catalysis.
Orthophosphate (P(i)) has two antagonistic effects on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), stimulation of activation and inhibition of catalysis by competition with the substrate RuBP. The enzyme binds P(i) at three distinct sites, two within the catalytic site (where 1P and 5P of ribulose 1,5-bisphosphate [RuBP] bind), and the third at the latch site (a positively charged pocket involved in active-site closure during catalysis). We examined the role of the latch and 5P sites in regulation of Rubisco activation and catalysis by introducing specific mutations in the enzyme of the cyanobacterium Synechocystis sp. strain PCC 6803. Whereas mutations at both sites abolished the P(i)-stimulated Rubisco activation, substitution of residues at the 5P site, but not at the latch site, affected the P(i) inhibition of Rubisco catalysis. Although some of these mutations substantially reduced the catalytic turnover of Rubisco and increased the K(m)(RuBP), they had little to moderate effect on the rate of photosynthesis and no effect on photoautotrophic growth. These findings suggest that in cyanobacteria, Rubisco does not limit photosynthesis to the extent previously estimated. These results indicate that both the latch and 5P sites participate in regulation of Rubisco activation, whereas P(i) binding only at the 5P site inhibits catalysis in a competitive manner.
Marcus
Yehouda
Y
Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel. yehoudam@post.tau.ac.il
Altman-Gueta
Hagit
H
Finkler
Aliza
A
Gurevitz
Michael
M
D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010710:Phosphates / Q000378:metabolism*; D010788:Photosynthesis / Q000235:genetics / Q000502:physiology; D011487:Protein Conformation; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry* / Q000378:metabolism*; D046939:Synechocystis / Q000201:enzymology* / Q000235:genetics
D010710:Phosphates; D012273:Ribulose-Bisphosphate Carboxylase