Rasche M E, Smith K S, Ferry J G
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802-4500, USA.
J Bacteriol. 1997 Dec;179(24):7712-7. doi: 10.1128/jb.179.24.7712-7717.1997.
Phosphotransacetylase catalyzes the following reaction: CoASH + CH3CO2PO3(2-) <==> CH3COSCoA + HPO4(2-) (where CoA is coenzyme A). Based on biochemical characterization of the enzyme from the obligate anaerobe Clostridium kluyveri, a ternary mechanism was proposed in which an unspecified cysteine abstracts a proton from CoASH forming a nucleophilic thiolate anion which attacks acetyl phosphate (J. Henkin and R. H. Abeles, Biochemistry 15:3472-3479, 1976). Heterologous production in Escherichia coli of the phosphotransacetylase from Methanosarcina thermophila, an obligately anaerobic methanoarchaeon, allowed site-specific replacements to identify essential residues. All four cysteines present in the sequence were individually replaced with alanine, and the kinetic constants of the altered enzymes were determined. The results indicated that only C159 is essential for activity; however, replacement with serine resulted in a fully active enzyme. Activity of the unaltered phosphotransacetylase was sensitive to N-ethylmaleimide. Inhibition kinetics of altered enzymes indicated that this sensitivity resulted from modification of C312, which is at the active site but itself is nonessential for catalysis. Five arginines were individually replaced with glutamine. Kinetic analysis of the altered enzymes identified R310 as essential for activity. Of the four nonessential for activity, R87 and R133 appear to be involved in binding CoA.
辅酶A（CoASH）+乙酰磷酸（CH3CO2PO3(2-)）⇌乙酰辅酶A（CH3COSCoA）+磷酸根离子（HPO4(2-)）（其中CoA为辅酶A）。基于对专性厌氧菌克氏梭菌中该酶的生化特性研究，提出了一种三元机制，即一个未明确的半胱氨酸从辅酶A上夺取一个质子，形成亲核硫醇盐阴离子，该阴离子攻击乙酰磷酸（J. Henkin和R. H. Abeles，《生物化学》15:3472 - 3479，1976）。嗜热甲烷八叠球菌（一种专性厌氧的甲烷古菌）的磷酸转乙酰酶在大肠杆菌中的异源表达，使得通过位点特异性替换来鉴定必需残基成为可能。序列中存在的所有四个半胱氨酸都分别被丙氨酸替换，并测定了改变后酶的动力学常数。结果表明，只有C159对活性至关重要；然而，用丝氨酸替换后得到了一种完全有活性的酶。未改变的磷酸转乙酰酶的活性对N - 乙基马来酰亚胺敏感。改变后酶的抑制动力学表明，这种敏感性是由活性位点处的C312被修饰所致，而C312本身对催化并非必需。五个精氨酸分别被谷氨酰胺替换。对改变后酶的动力学分析确定R310对活性至关重要。在对活性非必需的四个精氨酸中，R87和R133似乎参与辅酶A的结合。
