The carbamoyl-phosphate synthetase of Pyrococcus furiosus is enzymologically and structurally a carbamate kinase

Citation
M. Uriarte et al., The carbamoyl-phosphate synthetase of Pyrococcus furiosus is enzymologically and structurally a carbamate kinase, J BIOL CHEM, 274(23), 1999, pp. 16295-16303
Citations number
51
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
274
Issue
23
Year of publication
1999
Pages
16295 - 16303
Database
ISI
SICI code
0021-9258(19990604)274:23<16295:TCSOPF>2.0.ZU;2-Y
Abstract
The hyperthermophiles Pyrococcus furiosus and Pyrococcus abyssi make pyrimi dines and arginine from carbamoyl phosphate (CP) synthesized by an enzyme t hat differs from other carbamoyl-phosphate synthetases and that resembles c arbamate kinase (CK) in polypeptide mass, amino acid sequence, and oligomer ic organization, This enzyme was reported to use ammonia, bicarbonate, and two ATP molecules as carbamoyl-phosphate synthetases to make CP and to exhi bit bicarbonate-dependent ATPase activity. We have reexamined these finding s using the enzyme of P, furiosus expressed in Escherichia coil from the co rresponding gene cloned in a plasmid, We show that the enzyme uses chemical ly made carbamate rather than ammonia and bicarbonate and catalyzes a react ion with the stoichiometry and equilibrium that are typical for CK, Further more, the enzyme catalyzes actively full reversion of the CK reaction and e xhibits little bicarbonate-dependent ATPase, In addition, it cross-reacts w ith antibodies raised against CK from Enterococcus faecium, and its three-d imensional structure, judged by x-ray crystallography of enzyme crystals, i s very similar to that of CK, Thus, the enzyme is, in all respects other th an its function in vivo, a CK, Because in other organisms the function of C K is to make ATP from ADP and CP derived from arginine catabolism, this is the first example of using CK for making rather than using CP, The reasons for this use and the adaptation of the enzyme to this new function are disc ussed.