Zhao Y, Hawes J, Popov K M, Jaskiewicz J, Shimomura Y, Crabb D W, Harris R A
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
J Biol Chem. 1994 Jul 15;269(28):18583-7.
Regulation of the branched chain alpha-ketoacid dehydrogenase complex, the rate-limiting enzyme of branched chain amino acid catabolism, involves phosphorylation of 2 amino acid residues (site 1, serine 293; site 2, serine 303). To directly assess the roles played by these sites, site-directed mutagenesis was used to convert these serines to glutamates and/or alanines. Functional E1 heterotetramers were expressed in Escherichia coli carrying genes for E1 alpha and E1 beta under control of separate T7 promoters in a dicistronic vector. Mutation of phosphorylation site 1 serine to glutamate inactivated E1 activity, i.e. mimicked the effect of phosphorylation of site 1. Replacement of the site 1 serine with alanine greatly increased Km for the alpha-ketoacid substrate but had no effect on maximum velocity. The site 1 serine to alanine mutant was phosphorylated at site 2, but phosphorylation had no effect upon enzyme activity. Mutation of site 2 serine to either glutamate or alanine also had no effect upon enzyme activity, but phosphorylation of these proteins at site 1 inhibited enzyme activity. E1 mutated to change both phosphorylation site serines to glutamates was without enzyme activity. The binding affinity of E1 to the E2 core was not affected by mutation of the phosphorylation sites to glutamates, suggesting no gross perturbation of the association of E1 with the E2 core. The results provide direct evidence that a negative charge at phosphorylation site 1 is responsible for kinase-mediated inactivation of E1. Site 2 is silent with respect to regulation of activity by phosphorylation.
支链α-酮酸脱氢酶复合体是支链氨基酸分解代谢的限速酶，对其调节涉及2个氨基酸残基（位点1，丝氨酸293；位点2，丝氨酸303）的磷酸化。为了直接评估这些位点所起的作用，采用定点诱变将这些丝氨酸转化为谷氨酸和/或丙氨酸。功能性E1异源四聚体在大肠杆菌中表达，该大肠杆菌在双顺反子载体中，在单独的T7启动子控制下携带E1α和E1β的基因。将磷酸化位点1的丝氨酸突变为谷氨酸会使E1活性失活，即模拟了位点1磷酸化的效果。用丙氨酸取代位点1的丝氨酸会大大增加α-酮酸底物的Km，但对最大速度没有影响。位点1丝氨酸突变为丙氨酸的突变体在位点2被磷酸化，但磷酸化对酶活性没有影响。将位点2的丝氨酸突变为谷氨酸或丙氨酸对酶活性也没有影响，但这些蛋白质在位点1的磷酸化会抑制酶活性。突变为将两个磷酸化位点丝氨酸都变为谷氨酸的E1没有酶活性。将磷酸化位点突变为谷氨酸不会影响E1与E2核心的结合亲和力，这表明E1与E2核心的结合没有受到严重干扰。结果提供了直接证据，表明磷酸化位点1处的负电荷是激酶介导的E1失活的原因。位点2在通过磷酸化调节活性方面没有作用。
