Facilitation of the terminal proton transfer reaction of ribulose 1,5-bisphosphate carboxylase/oxygenase by active-site Lys166.
The terminal step in the carboxylation pathway catalyzed by ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is stereospecific protonation of the C-2 aci-acid of 3-phosphoglycerate (PGA). X-ray crystallographic results favor the epsilon-amino group of Lys166 as the proton donor in this step [Knight et al. (1990) J. Mol. Biol. 215, 113]. Nonetheless, position-166 mutants are able to catalyze forward processing of isolated 2-carboxy-3-ketoarabinitol 1,5-bisphosphate (CKABP), the carboxylated reaction intermediate [Lorimer G.H., & Hartman, F.C. (1988) J. Biol. Chem. 263, 6468]. Prior assays for intermediate processing relied solely on formation of acid-stable radioactivity from acid-labile [2'-14C]CKABP. Therefore, PGA, the normal reaction product, may not have been distinguished from pyruvate, the product from beta-elimination of phosphate from the terminal aci-acid intermediate [Andrews, T.J., & Kane, H.J. (1991) J. Biol. Chem. 266, 9447]. If Lys166 indeed serves as the terminal proton donor, mutants lacking an ionizable side chain at position 166 might process the carboxylated intermediate predominantly to pyruvate. We have thus used anion exchange chromatography and enzyme coupling to separate and identify the products from turnover of [2'-14C]CKABP by wild-type, K166G, and K166S enzymes. Although PGA is the only labeled product of significance formed by wild-type enzyme, pyruvate is a major labeled product formed by the mutants. These results provide the first direct functionally-based evidence that Lys166 is crucial to the last step in Rubisco-catalyzed conversion of RuBP to PGA.
Harpel
M R
MR
Biology Division, Oak Ridge National Laboratory, Tennessee 37831-8080, USA.
Hartman
F C
FC
D001665:Binding Sites / Q000235:genetics; D002852:Chromatography, Ion Exchange; D005988:Glyceric Acids / Q000737:chemistry / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010428:Pentosephosphates / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D011522:Protons; D019289:Pyruvic Acid / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013402:Sugar Alcohols / Q000737:chemistry / Q000378:metabolism
C102131:2-carboxy-3-ketoarabinitol 1,5-bisphosphate; D005988:Glyceric Acids; D010428:Pentosephosphates; D011522:Protons; D013402:Sugar Alcohols; C005156:3-phosphoglycerate; D019289:Pyruvic Acid; D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine