Multifaceted roles of Lys166 of ribulose-bisphosphate carboxylase/oxygenase as discerned by product analysis and chemical rescue of site-directed mutants.

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