A stable archaeal pyruvate carboxylase from the hyperthermophile Methanococcus jannaschii.
The pyruvate carboxylase (PYC) of the hyperthermophilic, strictly hydrogenotrophic, autotrophic and marine methanarchaeon Methanococcus jannaschii was purified to homogeneity. Optimal activity was at pH 8.5, > or = 80 degrees C, and a KCl concentration of 0.175 M. This enzyme is the most thermophilic PYC so far studied. Unlike the Methanobacterium thermoautotrophicum enzyme, Mc. jannaschii PYC was expressed in cells grown without an external source of biotin and in the purified form was stable during storage at 4, -20 and -80 degrees C. However, it was rapidly inactivated at 80 degrees C. The enzyme was insensitive to aspartate and glutamate, mildly inhibited by alpha-ketoglutarate, and was strongly inhibited by ATP and ADP (apparent Km, for ATP, 0.374 +/- 0.039 mM; apparent Ki for ATP, 5.34 +/- 2.14 mM; Ki for ADP, 0.89 +/- 0.18 mM). It was also strongly inhibited when the Mg2+ concentration in the assay exceeded that of ATP. Thus, this stable PYC could serve as a model for mechanistic studies on archaeal PYCs. It was apparently an alpha4beta4-type PYC composed of a non-biotinylated 55.5-kDa subunit (PYCA) and a 64.2-kDa biotinylated subunit (PYCB). The determined NH2-terminal sequences for these subunits provided additional support for our earlier proposal to rename the ORFs MJ1229 and MJ1231 in the NCBI Mc. jannaschii genome sequence database as PYCA and PYCB, respectively; even very recently, these have been misidentified as a subunit of acetyl-CoA carbxoylase (AccC) and the alpha-subunit of ion-pumping oxaloacetate decarboxylase (OADalpha), respectively.
Mukhopadhyay
B
B
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, 61801, USA. biswarup@life.uiuc.edu
Patel
V J
VJ
Wolfe
R S
RS
D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D001639:Bicarbonates / Q000378:metabolism / Q000494:pharmacology; D019921:Biotinylation; D002384:Catalysis; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D017017:Methanococcus / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011189:Potassium Chloride / Q000494:pharmacology; D020836:Protein Structure, Quaternary; D011766:Pyruvate Carboxylase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism / Q000494:pharmacology
D001639:Bicarbonates; D000244:Adenosine Diphosphate; D011189:Potassium Chloride; D019289:Pyruvic Acid; D000255:Adenosine Triphosphate; D011766:Pyruvate Carboxylase; D008274:Magnesium