Identification of the Allosteric Regulatory Site in Bacterial Phosphoribulokinase

Abstract

Bacterial phosphoribulokinases (PRKs) are octameric members of the adenylate kinase family of enzymes. The enzyme is allosterically activated by NADH and allosterically inhibited by AMP. We have determined the crystal structure of PRK from Rhodobacter sphaeroides bound to the ATP analogue AMP-PCP to a resolution of 2.6 A. The structure reveals that the ATP analogue does not bind to the canonical ATP site found in adenylate kinase family members. Rather, the AMP-PCP binds in two different orientations at the interface 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 221) is highly conserved in both prokaryotic and eukaryotic (nonallosterically regulated) PRKs, two (Arg 234 and Arg 257) are on a second subunit and conserved in only prokaryotic PRKs, and two (Arg 30 and Arg 31) are on a third subunit with only one of them (Arg 31) conserved in prokaryotic PRKs. Each 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 second subunit are directly involved in NADH binding, while the other arginines have a minimal effect on NADH binding. While the wild-type enzyme exhibits low maximal activity and hyperbolic kinetics with respect to ATP in the absence of NADH and high maximal activity and sigmoidal kinetics in the presence 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.

A RuBisCO-mediated carbon metabolic pathway in methanogenic archaea

“…1). Therefore, a low similarity in the C -terminal domain between MhPRK and RsPRK is consistent with differences in the manner of dimerization and quaternary structure of the two, which may lead to different enzymatic properties such as allosteric regulation of RsPRK (refs 23, 24), and not MhPRK by NADH and AMP.…”

Phosphoribulokinase: Current Perspectives on the Structure/Function Basis for Regulation and Catalysis

“…An independent approach that mated X-ray diffraction with directed mutagenesis was used by Harrison and colleagues (Kung et al, 1999). Taking advantage of the ability to crystallize PRK bound to the mononucleotide AMP-PCP, a 2.6 A structure of the binary complex was elucidated.…”

“…AMP-PCP, normally considered to be an ATP analog, did not bind where the ATP substrate is normally situated in nucleotide monophosphate kinase fold enzymes. Instead, the nucleotide bound at the interface between three subunits The figures depicts electron density attributable to the nucleotide analog 0, y-methylene ATP, which binds at the interface ofthree subunits ( Kung et al, 1999). Near the bound nucleotide are R221 from one subunit, R234 and R257 from a second subunit, and R30 and R3 1 from the third subunit.…”

“…Wild-type R. sphaeroides PRK will stoichiometrically bind ATP, the fluorescent alternative substrate trinitrophenyl ATP, or the spin-labeled analog S-acetamidoproxyl-ATPyS to form stable binary complexes. Such complex formation has been used to verify the integrity of catalytic mutants (Runquist et al, 1996 as well as effector site mutants (Kung et al, 1999). The test is based on the rationale that, if stoichiometric binding of analog reflects a structurally intact active site, the overall tertiary structure must exhibit a high level of integrity.…”

“…The stoichiometry of NADH binding has also been used to evaluate the structural integrity of catalytic and substrate binding mutants (Runquist et al, 1996. It has also allowed us to discriminate between mutants deficient in effector binding or deficient in transmission of the allosteric signal (Kung et al, 1999). Thus, the approach of mating spectroscopically active metabolite analogs with biophysical characterization of engineered proteins has demonstrated considerable utility and can be potentially applied to a wide variety of enzymes that bind nucleotide triphosphates (Potter and Miziorko, 1997) or other metabolites and their analogs (Narasimhan and Miziorko, 1997).…”