Channeling of Carbamoyl Phosphate to the Pyrimidine and Arginine Biosynthetic Pathways in the Deep Sea Hyperthermophilic Archaeon Pyrococcus abyssi

Cristina Purcarea,David R Evans,Guy Hervé

doi:10.1074/jbc.274.10.6122

Abstract

The kinetics of the coupled reactions between carbamoyl-phosphate synthetase (CPSase) and both aspartate transcarbamoylase (ATCase) and ornithine transcarbamoylase (OTCase) from the deep sea hyperthermophilic archaeon Pyrococcus abyssi demonstrate the existence of carbamoyl phosphate channeling in both the pyrimidine and arginine biosynthetic pathways. Isotopic dilution experiments and coupled reaction kinetics analyzed within the context of the formalism proposed by Ovádi et al. (Ovádi, J., Tompa, P., Vertessy, B., Orosz, F., Keleti, T., and Welch, G. R. (1989) Biochem. J. 257, 187-190) are consistent with a partial channeling of the intermediate at 37 degrees C, but channeling efficiency increases dramatically at elevated temperatures. There is no preferential partitioning of carbamoyl phosphate between the arginine and pyrimidine biosynthetic pathways. Gel filtration chromatography at high and low temperature and in the presence and absence of substrates did not reveal stable complexes between P. abyssi CPSase and either ATCase or OTCase. Thus, channeling must occur during the dynamic association of coupled enzymes pairs. The interaction of CPSase-ATCase was further demonstrated by the unexpectedly weak inhibition of the coupled reaction by the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA). The anomalous effect of PALA suggests that, in the coupled reaction, the effective concentration of carbamoyl phosphate in the vicinity of the ATCase active site is 96-fold higher than the concentration in the bulk phase. Channeling probably plays an essential role in protecting this very unstable intermediate of metabolic pathways performing at extreme temperatures.

Highlights

The kinetics of the coupled reactions between carbamoyl-phosphate synthetase (CPSase) and both aspartate transcarbamoylase (ATCase) and ornithine transcarbamoylase (OTCase) from the deep sea hyperthermophilic archaeon Pyrococcus abyssi demonstrate the existence of carbamoyl phosphate channeling in both the pyrimidine and arginine biosynthetic pathways
The progress curve measured at 70 °C for the purified P. abyssi CPSase coupled to E. coli ATCase gives a transient time of 23.9 s and a normalized steady state carbamoyl phosphate concentration of 1.04 ␮M
Since the kinetics of P. abyssi OTCase have not been previously investigated, a preliminary characterization of the enzyme was a prerequisite for the channeling studies

Summary

RESULTS

P. abyssi Ornithine Transcarbamoylase—The catalytic and regulatory properties of P. abyssi CPSase and ATCase were previously described [5, 24]. In the P. abyssi CPSase-ATCase-coupled reaction, dilution of the endogenous intermediate by high concentrations of exogenous carbamoyl phosphate demonstrates that under these conditions channeling is partial or leaky, not absolute. These experiments clearly show that in P. abyssi the endogenously synthesized intermediate does not freely equilibrate with carbamoyl phosphate in the bulk phase. CPSase-OTCase-coupled Reaction—The effect of exogenous carbamoyl phosphate on the incorporation of the radiolabeled intermediate into citrulline was measured at 37 °C in the P. abyssi cell-free extracts employing the same saturating concentrations of CPSase substrates and 0.2 mM ornithine (Fig. 1). Intermediate values for these parameters are indicative of partial or leaky channeling

Carbamoyl phosphate

Reaction t v

DISCUSSION