Multifaceted Roles of Lys166 of Ribulose-bisphosphate Carboxylase/Oxygenase As Discerned by Product Analysis and Chemical Rescue of Site-Directed Mutants
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- 作者：Mark R. Harpel ; Frank W. Larimer ; and Fred C. Hartman
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：January 29, 2002
- 年：2002
- 卷：41
- 期：4
- 页码：1390 - 1397
- 全文大小：110K
- 年卷期：v.41,no.4(January 29, 2002)
- ISSN：1520-4995
文摘
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 
-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 terminalaci-acid intermediate. As a further test of the postulate, the effects of subtle perturbation of the Lys166side chain on the carboxylation/oxygenation partitioning ratio (
) 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, for the aminoethylatedenzyme was increased by ~30%, and 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.
-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 terminalaci-acid intermediate. As a further test of the postulate, the effects of subtle perturbation of the Lys166side chain on the carboxylation/oxygenation partitioning ratio () 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, for the aminoethylatedenzyme was increased by ~30%, and 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.