Identification of the allosteric regulatory site in bacterial phosphoribulokinase

Bacterial phosphoribulokinases (PRKs) are octameric members of the adenylat e kinase family of enzymes. The enzyme is allosterically activated by NADH and allosterically inhibited by AMP. We have determined the crystal structu re of PRK from Rhodobacter sphaeroides bound to the ATP analogue AMP-PCP to a resolution of 2.6 Angstrom. The structure reveals that the ATP analogue does not bind to the canonical ATP site found in adenylate kinase family me mbers, Rather, the AMP-PCP binds in two different orientations at the inter face of three of the monomers in the octamer. This interface was previously characterized as having an unusually large number of arginine residues, Of the five arginine residues that are near the bound nucleotide, one (Arg 22 1) is highly conserved in both prokaryotic and eukaryotic (nonallostericall y regulated) PRKs, two (Arg 234 and Arg 257) are on a second subunit and co nserved in only prokaryotic PRKs, and two (Arg 30 and Arg 31) are on a thir d subunit with only one of them (Arg 31) conserved in prokaryotic PRKs. Eac h of these arginine residues was converted by site-directed mutagenesis to alanine. Fluorescence binding data suggest that none of these arginines are involved in active site ATP binding and that Arg 234 and Arg. 257 on the s econd subunit are directly involved in NADH binding, while the other argini nes have a minimal effect on NADH binding. While the wild-type enzyme exhib its low maximal activity and hyperbolic kinetics with respect to ATP in the absence of NADH and high maximal activity and sigmoidal kinetics in the pr esence of NADH, the R31A mutant exhibits identical hyperbolic kinetics with respect to ATP in the presence or absence of NADH, Thus, the transmission of allosteric information from one subunit to another is conducted through a single path that includes NADH and Arg 31.