Minard K I, McAlister-Henn L
Department of Biological Chemistry, College of Medicine, University of California, Irvine 92717.
Arch Biochem Biophys. 1994 Dec;315(2):302-9. doi: 10.1006/abbi.1994.1504.
The cytosolic isozyme of malate dehydrogenase, MDH2, was previously shown to be subject to rapid inactivation and proteolysis following the addition of glucose to yeast cultures growing on nonfermentable carbon sources. In this report, we show that MDH2 is phosphorylated during the process of glucose-induced degradation. A truncated active form of MDH2 lacking the first 12 residues of the amino terminus was previously found to be resistant to glucose-induced degradation and, as shown in this study, is not subject to phosphorylation. Site-directed mutagenesis was conducted to change Ser-12 in the authentic enzyme to Ala-12 and to Asp-12. The S12A substitution has little effect on glucose-induced phosphorylation and degradation, whereas the enzyme with the S12D substitution is subject to phosphorylation and inactivation but not to rapid degradation. This provides clear evidence that inactivation is not simply a result of degradation. Additional mutagenesis was conducted to change His-214, a critical active site residue, to Leu-214. Analysis of expression of full-length and truncated forms of the H214L enzyme demonstrated that catalytic inactivity is not a prerequisite for degradation and confirmed an essential role for the amino terminus of the authentic enzyme in this phenomenon.
苹果酸脱氢酶的胞质同工酶MDH2先前已表明，在向以非发酵碳源生长的酵母培养物中添加葡萄糖后，会迅速失活并发生蛋白水解。在本报告中，我们表明MDH2在葡萄糖诱导的降解过程中会发生磷酸化。先前发现一种截短的活性形式的MDH2，其氨基末端缺少前12个残基，对葡萄糖诱导的降解具有抗性，并且如本研究所示，不会发生磷酸化。进行了定点诱变，将天然酶中的Ser-12分别变为Ala-12和Asp-12。S12A替换对葡萄糖诱导的磷酸化和降解影响很小，而具有S12D替换的酶会发生磷酸化和失活，但不会迅速降解。这提供了明确的证据，表明失活不仅仅是降解的结果。还进行了额外的诱变，将关键活性位点残基His-214变为Leu-214。对H214L酶全长和截短形式的表达分析表明，催化无活性不是降解的先决条件，并证实了天然酶的氨基末端在这一现象中的重要作用。
