Characterization of the propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica: residue Lys592 is required for propionyl-AMP synthesis.
The propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica catalyzes the first step of propionate catabolism, i.e., the activation of propionate to propionyl-CoA. The PrpE enzyme was purified, and its kinetic properties were determined. Evidence is presented that the conversion of propionate to propionyl-CoA proceeds via a propionyl-AMP intermediate. Kinetic experiments demonstrated that propionate was the preferred acyl substrate (kcat/Km = 1644 mM(-1) x s(-1)). Adenosine 5'-propyl phosphate was a potent inhibitor of the enzyme, and inhibition kinetics identified a Bi Uni Uni Bi Ping Pong mechanism for the reaction catalyzed by the PrpE enzyme. Site-directed mutagenesis was used to change the primary sequence of the wild-type protein at positions G245A, P247A, K248A, K248E, G249A, K592A, and K592E. Mutant PrpE proteins were purified, and the effects of the mutations on enzyme activity were investigated. Both PrpEK592 mutant proteins (K592A and K592E) failed to convert propionate to propionyl-CoA, and plasmids containing these alleles of prpE failed to restore growth on propionate of S. enterica carrying null prpE alleles on their chromosome. Both PrpEK592 mutant proteins converted propionyl-AMP to propionyl-CoA, suggesting residue K592 played no discernible role in thioester bond formation. To the best of our knowledge, these mutant proteins are the first acyl-CoA synthetases reported that are defective in adenylation activity.
Horswill
Alexander R
AR
Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Escalante-Semerena
Jorge C
JC
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000096:biosynthesis* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000378:metabolism; D020816:Amino Acid Motifs / Q000235:genetics; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017124:Conserved Sequence / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011422:Propionates / Q000378:metabolism*; D019779:Salmonella enterica / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
D000214:Acyl Coenzyme A; D004791:Enzyme Inhibitors; D011422:Propionates; C009061:propionyl-coenzyme A; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; C068072:propionate - CoA ligase; D008239:Lysine