Ab initio calculations [King, W. A., et al. (1998)
Biochemistry 37, 15414-15422] of an active-site mimic of
D-ribulose-1,5-bisphosphate carboxylase/oxygenase suggest that active-site Lys166 plays arole in carboxylation in addition to its functions in the initial deprotonation and final protonation steps.To test this postulate, the turnover of 1-
3H-labeled
D-ribulose 1,5-bisphosphate (RuBP) by impaired position-166 mutants was characterized. Although these mutants catalyze slow enolization of RuBP, most of theRuBP-enediol undergoes
![](/images/gifchars/beta2.gif)
-elimination of phosphate to form 2,3-pentodiulose 5-phosphate, signifyingdeficiencies in normal carboxylation and oxygenation. Much of the remaining RuBP-enediol is carboxylatedbut forms pyruvate, rather than 3-phospho-
D-glycerate, due to incapacity in protonation of the terminal
aci-acid intermediate. As a further test of the postulate, the effects of subtle perturbation of the Lys166side chain on the carboxylation/oxygenation partitioning ratio (
![](/images/gifchars/tau.gif)
) were determined. To eliminate achemically reactive site, Cys58 was replaced by a seryl residue without any loss of activity. The virtuallyinactive K166C-C58S double mutant was chemically rescued by aminoethylation or aminopropylation toreinsert a lysyl-like side chain at position 166. Relative to the wild-type value,
![](/images/gifchars/tau.gif)
for the aminoethylatedenzyme was increased by ~30%, and
![](/images/gifchars/tau.gif)
for the aminopropylated enzyme was decreased by ~80%. Thus,two lines of experimentation support the theoretically based conclusion for the importance of Lys166 inthe reaction of RuBP-enediol with gaseous substrates.