Characterization of the aspartate transcarbamoylase from Methanococcus jannaschii.
The genes from the thermophilic archaeabacterium Methanococcus jannaschii that code for the putative catalytic and regulatory chains of aspartate transcarbamoylase were expressed at high levels in Escherichia coli. Only the M. jannaschii PyrB (Mj-PyrB) gene product exhibited catalytic activity. A purification protocol was devised for the Mj-PyrB and M. jannaschii PyrI (Mj-PyrI) gene products. Molecular weight measurements of the Mj-PyrB and Mj-PyrI gene products revealed that the Mj-PyrB gene product is a trimer and the Mj-PyrI gene product is a dimer. Preliminary characterization of the aspartate transcarbamoylase from M. jannaschii cell-free extract revealed that the enzyme has a similar molecular weight to that of the E. coli holoenzyme. Kinetic analysis of the M. jannaschii aspartate transcarbamoylase from the cell-free extract indicates that the enzyme exhibited limited homotropic cooperativity and little if any regulatory properties. The purified Mj-catalytic trimer exhibited hyperbolic kinetics, with an activation energy similar to that observed for the E. coli catalytic trimer. Homology models of the Mj-PyrB and Mj-PyrI gene products were constructed based on the three-dimensional structures of the homologous E. coli proteins. The residues known to be critical for catalysis, regulation, and formation of the quaternary structure from the well characterized E. coli aspartate transcarbamoylase were compared.
Hack
E S
ES
Department of Chemistry, Boston College, Merkert Chemistry Center, Chestnut Hill, Massachusetts 02467, USA.
Vorobyova
T
T
Sakash
J B
JB
West
J M
JM
Macol
C P
CP
Hervé
G
G
Williams
M K
MK
Kantrowitz
E R
ER
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry*; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D017017:Methanococcus / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D001221:Aspartate Carbamoyltransferase