Kinetics and reaction mechanism of the carbamylphosphate synthetase of a multienzyme aggregate from yeast.
We have studied the kinetics and reaction mechanism of the carbamylphosphate synthetase of an enzyme aggregate functioning in the pyrimidine pathway of yeast. MG--ATP was found to be one of the substrates of the enzyme reaction which was activated by free Mg-2+ and inhibited by free ATP. Feedback inhibition by UTP was non-competitive with respect to both glutamine and bicarbonate. Potassium ions were essential for activity and could not be replaced by sodium. Glutamine could be replaced partially by ammonium ions as nitrogen donor. A bicarbonate-dependent cleavage of ATP was shown to take place in the absence of L-glutamine; L-glutamate was a competitive inhibitor of L-glutamine and the enzyme was shown to synthesize ATP when incubated with ADP and carbamyl phosphate. The reaction mechanism was found to involve sequential addition of the substrates bicarbonate and Mg--ATP and release of ADP, followed by addition of the third substrate glutamine. The purine nucleotide XMP had a pronounced activating effect on the enzyme, acting at a site different from that of UTP. Saturating levels of Mg--ATP eliminated this activation.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000494:pharmacology; D000641:Ammonia / Q000378:metabolism; D001639:Bicarbonates / Q000494:pharmacology; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000378:metabolism*; D002221:Carbamyl Phosphate / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005973:Glutamine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008956:Models, Chemical; D009097:Multienzyme Complexes / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014500:Uracil Nucleotides / Q000494:pharmacology; D014970:Xanthines / Q000494:pharmacology
D001639:Bicarbonates; D009097:Multienzyme Complexes; D014500:Uracil Nucleotides; D014970:Xanthines; D005973:Glutamine; D002221:Carbamyl Phosphate; D000244:Adenosine Diphosphate; D000641:Ammonia; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D008274:Magnesium; D011188:Potassium