Characterization of the propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica:: Residue Lys592 is required for propionyl-AMP synthesis
Characterization of the propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica:: Residue Lys592 is required for propionyl-AMP synthesis
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DOI：
10.1021/bi015647q
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发表时间：
2002-02-19
期刊：
影响因子：
2.9
通讯作者：
Escalante-Semerena, JC
中科院分区：
文献类型：
--
作者：
Horswill, AR;Escalante-Semerena, JC
肠沙门氏菌的丙酰辅酶a合成酶（PrpE）酶催化丙酸分解代谢的第一步，即丙酸活化为丙酰辅酶a。纯化了PrpE酶，并测定了其动力学性质。证据表明丙酸转化为丙酰辅酶a是通过丙酰- amp中间体进行的。动力学实验表明丙酸盐是首选的酰基底物(k(cat)/ k -m = 1644 mM（-1) s(-1)）。磷酸腺苷5′-丙基是一种有效的酶抑制剂，抑制动力学鉴定了PrpE酶催化反应的Bi Uni Uni Bi乒乓机制。利用定点诱变技术改变野生型蛋白G245A、P247A、K248A、K248E、G249A、K592A和K592E位点的初级序列。纯化了突变体PrpE蛋白，并研究了突变对酶活性的影响。PrpEK592突变蛋白（K592A和K592E）都不能将丙酸转化为丙酰辅酶a，并且含有这些prpE等位基因的质粒不能恢复染色体上携带空prpE等位基因的肠链球菌在丙酸上的生长。两个PrpEK592突变蛋白都将丙酰- amp转化为丙酰-辅酶a，这表明残基K592在硫酯键形成中没有明显的作用。据我们所知，这些突变蛋白是首次报道的酰基辅酶a合成酶在腺苷化活性上存在缺陷。
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 (k(cat)/K-m = 1644 mM(-1) 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.