Enzymatic characterization of AMP phosphorylase and ribose-1,5-bisphosphate isomerase functioning in an archaeal AMP metabolic pathway

Abstract

AMP phosphorylase (AMPpase), ribose-1,5-bisphosphate (R15P) isomerase, and type III ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been proposed to constitute a novel pathway involved in AMP metabolism in the Archaea. Here we performed a biochemical examination of AMPpase and R15P isomerase from Thermococcus kodakarensis. R15P isomerase was specific for the α-anomer of R15P and did not recognize other sugar compounds. We observed that activity was extremely low with the substrate R15P alone but was dramatically activated in the presence of AMP. Using AMP-activated R15P isomerase, we reevaluated the substrate specificity of AMPpase. AMPpase exhibited phosphorylase activity toward CMP and UMP in addition to AMP. The [S]-v plot (plot of velocity versus substrate concentration) of the enzyme toward AMP was sigmoidal, with an increase in activity observed at concentrations higher than approximately 3 mM. The behavior of the two enzymes toward AMP indicates that the pathway is intrinsically designed to prevent excess degradation of intracellular AMP. We further examined the formation of 3-phosphoglycerate from AMP, CMP, and UMP in T. kodakarensis cell extracts. 3-Phosphoglycerate generation was observed from AMP alone, and from CMP or UMP in the presence of dAMP, which also activates R15P isomerase. 3-Phosphoglycerate was not formed when 2-carboxyarabinitol 1,5-bisphosphate, a Rubisco inhibitor, was added. The results strongly suggest that these enzymes are actually involved in the conversion of nucleoside monophosphates to 3-phosphoglycerate in T. kodakarensis.

Fig 1

Effects of various compounds on the activity of R15P isomerase. (A) R15P isomerase activity was measured by the enzyme coupling method with enzymatically prepared or chemically synthesized R15P (E-R15P or C-R15P, respectively). With C-R15P, the effects of the following compounds were examined: 3 mM AMP, 18 mM sodium phosphate (P_i), 0.4 mM adenine, 3 mM CMP, 3 mM GMP, 3 mM UMP, 3 mM TMP, or 0.4 mM NAD⁺. (B) Activation of R15P isomerase in the presence of varying concentrations of AMP or GMP. The initial velocities of R15P isomerase measured with coupling enzymes in the presence of AMP (circles) or GMP (triangles) are shown. (C) Activation of R15P isomerase in the presence of various compounds with an adenosyl moiety. The initial velocities of R15P isomerase were measured with coupling enzymes at varying concentrations of the following compounds: AMP (filled diamonds), dAMP (filled triangles), methylthioadenosine (filled circles), adenosine (plus signs), S-adenosylhomocysteine (filled squares), S-adenosylmethionine (asterisks), or ATP (open circles). AMP (open diamonds) and ADP (open squares) data are the results of measurements by HPLC. (D) Results of AMP and ADP measurements by HPLC in panel C, up to 2.5 mM. In all experiments, C-R15P was used at a concentration of 5 mM.

Fig 2

Kinetic analysis of R15P isomerase. Initial velocities of R15P isomerase were measured with coupling enzymes in the presence of varying concentrations of C-R15P. AMP (10 mM) was present for all measurements.

Fig 3

Kinetic analyses of AMPpase. (A) Kinetic analysis of AMPpase activities toward NMPs. Initial velocities of AMPpase were measured with coupling enzymes in the presence of varying concentrations of AMP (circles), CMP (squares), GMP (triangles), or UMP (diamonds). The concentration of P_i was constant at 20 mM. (B) Enlarged view of the results shown in panel A. (C) Kinetic analysis of AMPpase activity toward dCMP. Initial velocities of AMPpase were measured in the presence of varying concentrations of dCMP (triangles), AMP (circles), or CMP (squares). The results for CMP were obtained by both the enzyme coupling method (open symbols) and HPLC (filled symbols). The concentration of P_i was constant at 20 mM. (D) Dependence of AMPpase activity on P_i concentrations. Initial velocities were measured in the presence of varying concentrations of P_i and 20 mM AMP.

Fig 4

Protein levels of AMPpase, R15P isomerase, and Rubisco in T. kodakarensis cells. Western blot analyses using an antiserum containing polyclonal anti-AMPpase, anti-R15P isomerase, or anti-Rubisco antibodies were performed against extracts of T. kodakarensis cells grown in ASW-YT-Pyr or ASW-YT-Pyr-Nuc medium.

Fig 5

Substrate specificities and regulatory properties of AMPpase, R15P isomerase, and Rubisco in T. kodakarensis.