Characterization of the Propionyl-CoA Synthetase (PrpE) Enzyme of Salmonella enterica: Residue Lys592 Is Required for Propionyl-AMP Synthesis

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• 作者：Alexander R. Horswill and Jorge C. Escalante-Semerena
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：February 19, 2002
• 年：2002
• 卷：41
• 期：7
• 页码：2379 - 2387
• 全文大小：102K
• 年卷期：v.41,no.7(February 19, 2002)
• ISSN：1520-4995

文摘

The propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica catalyzes the first stepof 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 topropionyl-CoA proceeds via a propionyl-AMP intermediate. Kinetic experiments demonstrated thatpropionate was the preferred acyl substrate (k_cat/K_m = 1644 mM^-1 s^-1). Adenosine 5'-propyl phosphatewas a potent inhibitor of the enzyme, and inhibition kinetics identified a Bi Uni Uni Bi Ping Pongmechanism for the reaction catalyzed by the PrpE enzyme. Site-directed mutagenesis was used to changethe 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 activitywere investigated. Both PrpEK592 mutant proteins (K592A and K592E) failed to convert propionate topropionyl-CoA, and plasmids containing these alleles of prpE failed to restore growth on propionate ofS. enterica carrying null prpE alleles on their chromosome. Both PrpEK592 mutant proteins convertedpropionyl-AMP to propionyl-CoA, suggesting residue K592 played no discernible role in thioester bondformation. To the best of our knowledge, these mutant proteins are the first acyl-CoA synthetases reportedthat are defective in adenylation activity.