THE KINETICS OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE IN-VIVO INFERRED FROM MEASUREMENTS OF PHOTOSYNTHESIS IN LEAVES OF TRANSGENIC TOBACCO/

Transgenic tobacco (Nicotiana tabacum L. cv. W38) with an antisense ge ne directed against the mRNA of the ribulose-1,5-bisphosphate carboxyl ase/oxygenase (Rubisco) small subunit was used to determine the kineti c properties of Rubisco in vivo. The leaves of these plants contained only 34% as much Rubisco as those of the wild type, but other photosyn thetic components were not significantly affected. Consequently, the r ate of CO2 assimilation by the antisense plants was limited by Rubisco activity over a wide range of CO2 partial pressures. Unlike in the wi ld-type leaves, where the rate of regeneration of ribulose bisphosphat e limited CO2 assimilation at intercellular partial pressures above 40 0 mu bar, photosynthesis in the leaves of the antisense plants respond ed hyperbolically to CO2, allowing the kinetic parameters of Rubisco i n vivo to be inferred. We calculated a maximal catalytic turnover rate , k(cat), of 3.5+/-0.2 mol CO2.(mol sites)(-1).s(-1) at 25 degrees C i n vivo. By comparison, we measured a value of 2.9 mol CO2.(mol sites)( -1).s(-1) in vitro with leaf extracts. To estimate the Michaelis-Mente n constants for CO2 and O-2, the rate of CO2 assimilation was measured at 25 degrees C at different intercellular partial pressures of CO2 a nd O-2. These measurements were combined with carbon-isotope analysis (C-13/C-12) of CO2 in the air passing over the leaf to estimate the co nductance for transfer of CO2 from the substomatal cavities to the sit es of carboxylation (0.3 mol m(-2).s(-1).bar(-1)) and thus the partial pressure of CO2 at the sites of carboxylation. The calculated Michael is-Menten constants for CO2 and O-2 were 259+/-57 mu bar (8.6+/-1.9 mu M) and 179 mbar (226 mu M), respectively, and the effective Michaelis -Menten constant for CO2 in 200 mbar O-2 was 549 mu bar (18.3 mu M). F rom measurements of the photocompensation point (Gamma = 38.6 mu bar) we estimated Rubisco's relative specificity for CO2, as opposed to O- 2 to be 97.5 in vivo. These values were dependent on the size of the e stimated CO2-transfer conductance.