Kinetic characterization of human phosphopantothenoylcysteine synthetase.
Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the formation of phosphopantothenoylcysteine from (R)-phosphopantothenate and L-cysteine with the concomitant consumption of a nucleotide triphosphate. Herein, the human coaB gene encoding PPCS is cloned into pET23a and overexpressed in E. coli BL21(DE3), to yield 10mg of purified enzyme per liter of culture. Detailed kinetic studies found that this PPCS follows a similar Bi Uni Uni Bi Ping Pong mechanism as previously described for the E. faecalis PPCS, except that the human enzyme can use both ATP and CTP with similar affinity. One significant difference for human PPCS catalysis with respect to ATP and CTP is that the enzyme shows cooperative binding of ATP, measured as a Hill constant of 1.7. PPCS catalysis under CTP conditions displayed Michaelis constants of 265 microM, 57 microM, and 16 microM for CTP, PPA, and cysteine, respectively, with a kcat of 0.53+/-0.01 s(-1) for the reaction. Taking into account the cooperativity under ATP condition, PPCS exhibited Michaelis constants of 269 microM, 13 microM, and 14 microM for ATP, PPA, and cysteine, respectively, with a kcat of 0.56 s(-1) for the reaction. Oxygen transfer studies found that 18O from [carboxyl-18O] phosphopantothenate is incorporated into the AMP or CMP produced during PPCS catalysis, consistent with the formation of a phosphopantothenoyl cytidylate or phosphopantothenoyl adenylate intermediate, supporting similar catalytic mechanisms under both CTP and ATP conditions. Inhibition studies with GTP and UTP as well as product inhibition studies with CMP and AMP suggest that human PPCS lacks strong nucleotide selectivity.
Yao
Jiangwei
J
Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065, USA.
Dotson
Garry D
GD
D000255:Adenosine Triphosphate / Q000378:metabolism; D003570:Cytidine Triphosphate / Q000378:metabolism; D013293:Enterococcus faecalis / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010453:Peptide Synthases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D014544:Uridine Triphosphate / Q000494:pharmacology
D004791:Enzyme Inhibitors; D008670:Metals; D011994:Recombinant Proteins; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C022374:phosphopantothenoyl-cysteine synthetase; D010100:Oxygen; D014544:Uridine Triphosphate