Jordan-Starck T C, Rodwell V W
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
J Biol Chem. 1989 Oct 25;264(30):17919-23.
Each of the four identical subunits of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase contains two cysteine residues, Cys156 and Cys296 (Beach, M. J., and Rodwell, V. W. (1989) J. Bacteriol. 171, 2994-3001). Both are accessible to modification by sulfhydryl reagents under nondenaturing conditions (Jordan-Starck, T. C., and Rodwell, V. W. (1989) J. Biol. Chem. 264, 17913-17918). We used site-directed mutagenesis to construct three mutant enzymes in which alanine replaced either or both cysteine residues. Mutant enzymes C156A, C296A, and C156/296A were over-expressed in Escherichia coli and were found to be fully active. Following their purification, all four forms of the enzyme were compared with respect to their catalytic efficiency, their affinities for the substrates of all four catalyzed reactions, and for their sensitivity to inactivation by sulfhydryl reagents. Replacement of cysteine residues with alanine residues had no major effect on either the specific activity or the affinity of the enzymes for any substrate. The mutants catalyzed all four HMG-CoA reductase reactions as efficiently as did the wild-type enzyme, and coenzyme A stimulated mevaldehyde reduction to the same extent as for wild-type HMG-CoA reductase. Mutant C156A and the cysteine-free mutant C156/296A were not inactivated by 5,5'-dithiobis(2-nitrobenzoate). By contrast, mutant C296A was inactivated to the same extent as was the wild-type enzyme. Following treatment of the mutant enzymes with N-ethylmaleimide, the four reductase reactions catalyzed by mutant C296A were inactivated to the same extent as for the wild-type enzyme. Neither mutant C156A nor C156/296A was affected by this reagent. We conclude that the sulfhydryl reagent-reactive group whose derivatization leads to loss of enzymatic activity is Cys156. However, this residue is not an essential active site residue since neither substrate binding nor catalysis was affected when it was replaced by alanine. Possible roles of cysteine in maintaining structural stability are discussed.
嗜甲基假单胞菌3-羟基-3-甲基戊二酰（HMG）-CoA还原酶的四个相同亚基中的每一个都含有两个半胱氨酸残基，即Cys156和Cys296（比奇，M. J.，和罗德韦尔，V. W.（1989年）《细菌学杂志》171，2994 - 3001）。在非变性条件下，二者都可被巯基试剂修饰（乔丹 - 斯塔克，T. C.，和罗德韦尔，V. W.（1989年）《生物化学杂志》264，17913 - 17918）。我们利用定点诱变构建了三种突变酶，其中丙氨酸取代了一个或两个半胱氨酸残基。突变酶C156A、C296A和C156/296A在大肠杆菌中过量表达，并且发现它们具有完全活性。纯化后，比较了该酶的所有四种形式在催化效率、对所有四种催化反应底物的亲和力以及对巯基试剂失活的敏感性方面的差异。用丙氨酸残基取代半胱氨酸残基对酶的比活性或对任何底物的亲和力都没有重大影响。这些突变体催化所有四种HMG - CoA还原酶反应的效率与野生型酶相同，并且辅酶A刺激甲羟醛还原的程度与野生型HMG - CoA还原酶相同。突变体C156A和不含半胱氨酸的突变体C156/296A不会被5,5'-二硫代双（2 - 硝基苯甲酸）失活。相比之下，突变体C296A的失活程度与野生型酶相同。用N - 乙基马来酰亚胺处理突变酶后，突变体C296A催化的四种还原酶反应失活程度与野生型酶相同。突变体C156A和C156/296A均不受该试剂影响。我们得出结论，其衍生化导致酶活性丧失的巯基试剂反应性基团是Cys156。然而，该残基不是必需的活性位点残基，因为当它被丙氨酸取代时，底物结合和催化都没有受到影响。讨论了半胱氨酸在维持结构稳定性方面可能的作用。
