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Multiple catalytic roles of His 287 of Rhodospirillum rubrum ribulose 1,5‐bisphosphate carboxylase/oxygenase
Author(s) -
Harpel Mark R.,
Larimer Frank W.,
Hartman Fred C.
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560070322
Subject(s) - rhodospirillum rubrum , pyruvate carboxylase , chemistry , oxygenase , rubisco , decarboxylation , carboxylation , stereochemistry , biochemistry , mutant , ribulose , active site , enzyme , catalysis , gene
Active‐site His 287 of Rhodospirillum rubrum ribulose 1,5‐bisphosphate (RuBP) carboxylase/oxygenase interacts with the C3‐hydroxyl of bound substrate or reaction‐intermediate analogue (CABP), water molecules, and ligands for the activator metal‐ion (Andersson I, 1996, J Mol Biol 259 :160‐174; Taylor TC, Andersson I, 1997, J Mol Biol 265 :432‐444). To test structure‐based postulates of catalytic functionality, His 287 was replaced with Asn or Gln. The mutants are not affected adversely in subunit assembly, activation (binding of Mg 2+ and carbamylation of Lys 191), or recognition of phosphorylated ligands; they bind CABP with even greater tenacity than does wild‐type enzyme. H287N and H287Q are severely impaired in catalyzing overall carboxylation (∼10 3 ‐fold and >10 5 ‐fold, respectively) and enolization (each mutant below threshold for detection) of RuBP. H287N preferentially catalyzes decarboxylation of carboxylated reaction intermediate instead of forward processing to phosphoglycerate. Analysis of RuBP turnover that occurs at high concentrations of mutants over extended time periods reveal > 10‐fold reduced CO 2 /O 2 specificities, elevated misprotonation of the enediol intermediate, and misprocessing of the oxygenated intermediate of the oxygenase pathway. These results are consistent with multifaceted roles for His 287 in promoting enediol formation, enediol tautomerization, and forward‐processing of carboxylated intermediate.