Multifaceted roles of Lys166 of ribulose-bisphosphate carboxylase/oxygenase as discerned by product analysis and chemical rescue of site-directed mutants.
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 a role in carboxylation in addition to its functions in the initial deprotonation and final protonation steps. To test this postulate, the turnover of 1-(3)H-labeled D-ribulose 1,5-bisphosphate (RuBP) by impaired position-166 mutants was characterized. Although these mutants catalyze slow enolization of RuBP, most of the RuBP-enediol undergoes beta-elimination of phosphate to form 2,3-pentodiulose 5-phosphate, signifying deficiencies in normal carboxylation and oxygenation. Much of the remaining RuBP-enediol is carboxylated but 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 Lys166 side chain on the carboxylation/oxygenation partitioning ratio (tau) were determined. To eliminate a chemically reactive site, Cys58 was replaced by a seryl residue without any loss of activity. The virtually inactive K166C-C58S double mutant was chemically rescued by aminoethylation or aminopropylation to reinsert a lysyl-like side chain at position 166. Relative to the wild-type value, tau for the aminoethylated enzyme was increased by approximately 30%, and tau for the aminopropylated enzyme was decreased by approximately 80%. Thus, two lines of experimentation support the theoretically based conclusion for the importance of Lys166 in the reaction of RuBP-enediol with gaseous substrates.
Harpel
Mark R
MR
Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA.
Larimer
Frank W
FW
Hartman
Fred C
FC
D000478:Alkylation; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D007525:Isoelectric Focusing; D008239:Lysine / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine