Aquifex aeolicus aspartate transcarbamoylase, an enzyme specialized for the efficient utilization of unstable carbamoyl phosphate at elevated temperature.
Aquifex aeolicus, an organism that flourishes at 95 degrees C, is one of the most thermophilic eubacteria thus far described. The A. aeolicus pyrB gene encoding aspartate transcarbamoylase (ATCase) was cloned, overexpressed in Escherichia coli, and purified by affinity chromatography to a homogeneous form that could be crystallized. Chemical cross-linking and size exclusion chromatography showed that the protein was a homotrimer of 34-kDa catalytic chains. The activity of A. aeolicus ATCase increased dramatically with increasing temperature due to an increase in kcat with little change in the Km for the substrates, carbamoyl phosphate and aspartate. The Km for both substrates was 30-40-fold lower than the corresponding values for the homologous E. coli ATCase catalytic subunit. Although rapidly degraded at high temperature, the carbamoyl phosphate generated in situ by A. aeolicus carbamoyl phosphate synthetase (CPSase) was channeled to ATCase. The transient time for carbamoyl aspartate formation was 26 s, compared with the much longer transient times observed when A. aeolicus CPSase was coupled to E. coli ATCase. Several other approaches provided strong evidence for channeling and transient complex formation between A. aeolicus ATCase and CPSase. The high affinity for substrates combined with channeling ensures the efficient transfer of carbamoyl phosphate from the active site of CPSase to that of ATCase, thus preserving it from degradation and preventing the formation of toxic cyanate.
Purcarea
Cristina
C
Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Ahuja
Anupama
A
Lu
Tun
T
Kovari
Ladislau
L
Guy
Hedeel I
HI
Evans
David R
DR
D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000235:genetics*; D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D003432:Cross-Linking Reagents; D001221:Aspartate Carbamoyltransferase; D004259:DNA-Directed DNA Polymerase