Pang S S, Duggleby R G
Centre for Protein Structure, Function and Engineering, Department of Biochemistry, The University of Queensland, Brisbane, Australia.
Biochemistry. 1999 Apr 20;38(16):5222-31. doi: 10.1021/bi983013m.
Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) catalyzes the first step in the biosynthesis of the branched-chain amino acids. In bacteria, the enzyme has a large subunit containing the catalytic machinery and a small subunit with a regulatory role. In eucaryotes, the evidence for a regulatory subunit is largely indirect and circumstantial. We investigated the possibility that the yeast open reading frame YCL009c is an AHAS small subunit. Analysis of the DNA sequence shows that it contains all the appropriate transcription, translation and regulatory signals. YCL009c was shown to be expressed in yeast and the protein localized in mitochondria where it undergoes removal of a transit peptide targeting sequence. This putative small subunit protein (ilv6) and the catalytic subunit of yeast AHAS (ilv2) were each overexpressed in Escherichia coli and purified to near homogeneity. Reconstitution studies showed that the ilv6 protein stimulates the catalytic activity of the ilv2 protein by up to 7-fold (from 6.8 +/- 0.7 to 49.0 +/- 1.8 U/mg) and confers upon it sensitivity to inhibition by valine (Ki = 0.16 +/- 0.02 mM). Valine inhibition is partially reversed by ATP. The reconstitution is favored by high concentrations of potassium phosphate ( approximately 1 M) and at neutral pH. Under optimal conditions for reconstitution, a dissociation constant for the subunits of 70 +/- 7 nM was determined. Valine inhibition is partial, resulting in a specific activity that is similar to that of the ilv2 protein alone. However, measurements of the Km for substrate rule out the possibility that valine inhibition is accomplished by dissociation of the subunits.
乙酰羟酸合酶（AHAS，EC 4.1.3.18）催化支链氨基酸生物合成的第一步。在细菌中，该酶有一个包含催化机制的大亚基和一个起调节作用的小亚基。在真核生物中，存在调节亚基的证据很大程度上是间接的和 circumstantial（此处可能有误，推测为“间接的和旁证的”）。我们研究了酵母开放阅读框YCL009c是AHAS小亚基的可能性。对DNA序列的分析表明，它包含所有适当的转录、翻译和调节信号。YCL009c在酵母中表达，并且该蛋白质定位于线粒体，在那里它经历靶向序列转运肽的去除。这种假定的小亚基蛋白（ilv6）和酵母AHAS的催化亚基（ilv2）各自在大肠杆菌中过表达并纯化至接近均一性。重组研究表明，ilv6蛋白可将ilv2蛋白的催化活性提高多达7倍（从6.8±0.7提高到49.0±1.8 U/mg），并使其对缬氨酸抑制敏感（Ki = 0.16±0.02 mM）。缬氨酸抑制可被ATP部分逆转。高浓度的磷酸钾（约1 M）和中性pH有利于重组。在重组的最佳条件下，测定亚基的解离常数为70±7 nM。缬氨酸抑制是部分性的，导致比活性与单独的ilv2蛋白相似。然而，对底物Km的测量排除了缬氨酸抑制是通过亚基解离实现的可能性。
