Functional analysis of the putative catalytic bases His-321 and Ser-368 of Rhodospirillum rubrum ribulose bisphosphate carboxylase/oxygenase by site-directed mutagenesis.
Numerous candidates have been suggested according to chemical and structural criteria for the active site base of ribulose bisphosphate carboxylase/oxygenase that catalyzes substrate enolization. We evaluate the functional significance of two such candidates, His-321 and Ser-368 of the Rhodospirillum rubrum enzyme, by site-directed mutagenesis. Position 321 mutants retain 3-12% of wild-type rates of both overall carboxylation and the initial enolization, with little effect on Km for CO2 or ribulose bisphosphate. Position 368 mutants exhibit approximately 1% of wild-type carboxylation but 4-9% of enolization, also accompanied by little effect on Km values. The modest catalytic facilitations elicited by these residues are incompatible with either acting as the crucial base. The enhanced efficiency of the position 368 mutants in enolization versus carboxylation clearly indicates that Ser-368 effects catalysis preferentially beyond the point of proton abstraction. Both sets of mutants bind the reaction intermediate analogue, 2-carboxy-D-arabinitol bisphosphate, stoichiometrically. Ligand exchange from complexes with position 321 mutants is increased relative to wild type, whereas complexes with position 368 mutants are more exchange-inert. Therefore, His-321 may assist stabilization of the transition state mimicked by the analogue.
Harpel
M R
MR
Protein Engineering Program, Oak Ridge National Laboratory, Tennessee 37831-8077.
Larimer
F W
FW
Hartman
F C
FC
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006639:Histidine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*
D012694:Serine; D006639:Histidine; D012273:Ribulose-Bisphosphate Carboxylase