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Fungal aspartate kinase mechanism and inhibition.

机译：真菌天门冬氨酸激酶的机制和抑制作用。

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摘要

Aspartate kinase (AK) from Saccharomyces cerevisiae (AKSc) catalyzes the first step in the aspartate pathway responsible for biosynthesis of L-threonine, L-isoleucine, and L-methionine in fungi. Little was known about amino acids important for AKSc substrate binding and catalysis. Hypotheses about important amino acids were tested using site directed mutagenesis to substitute these amino acids with others having different properties. Steady state kinetic parameters and pH titrations of the variant enzymes showed AKSc-K18 and H292 to be important for binding and catalysis.;Little was known about how the S. cerevisiae aspartate pathway kinases, AKSc and homoserine kinase (HSKSc), catalyze the transfer of the gamma-phosphate from adenosine triphosphate (ATP) to L-aspartate or L-homoserine, respectively. Two transfer paths are possible as are a range of mechanisms, with two extremes. Both kinases were shown to directly transfer the gamma-phosphate from ATP to the amino acid. Experimental evidence was consistent with an associative mechanism of phosphoryl transfer but did not rule out a dissociative mechanism.;AKSc and HSKSc are good targets for inhibitors because they catalyze phosphoryl transfers between very similar substrates. An attempt was made to rationally design inhibitors to these kinases by linking the substrates with a variable length linker to create bisubstrate compounds. This strategy failed inhibit AKSc and HSKSc, however, one of the bisubstrate compounds was a good inhibitor of AKIII from Escherichia coli.;Inhibition of any of the enzymes in the aspartate pathway would lead to reduced production of amino acids. A pathway assay was optimized to allow screening of chemical libraries in the hope of identifying inhibitors to the first four pathway enzymes. A high throughput screen of 1,000 compounds identified two compounds capable of inhibiting the assay, one of which was the best inhibitor identified for AKSc.
机译：来自酿酒酵母（AKSc）的天冬氨酸激酶（AK）催化负责真菌中L-苏氨酸，L-异亮氨酸和L-蛋氨酸生物合成的天冬氨酸途径的第一步。对于AKSc底物结合和催化重要的氨基酸知之甚少。使用定点诱变测试重要氨基酸的假设，以将这些氨基酸替换为其他具有不同特性的氨基酸。变体酶的稳态动力学参数和pH滴定表明AKSc-K18和H292对于结合和催化很重要。从三磷酸腺苷（ATP）到L-天门冬氨酸或L-高丝氨酸的γ-磷酸。两条机制都可能有两条转移路径，但有两个极端。两种激酶均显示可将γ-磷酸酯直接从ATP转移至氨基酸。实验证据与磷酸基转移的缔合机制一致，但并不排除解离机制。AKSc和HSKSc是抑制剂的良好靶标，因为它们催化非常相似的底物之间的磷酰基转移。尝试通过将底物与可变长度的接头连接以产生双底物化合物来合理设计这些激酶的抑制剂。该策略未能抑制AKSc和HSKSc，但是，双底物化合物之一是大肠杆菌AKIII的良好抑制剂。;天冬氨酸途径中任何酶的抑制都会导致氨基酸产生的减少。优化了途径测定法以允许筛选化学文库，以期希望鉴定出对前四种途径酶的抑制剂。通过对1,000种化合物的高通量筛选，鉴定出了两种能够抑制测定的化合物，其中一种是确定为AKSc的最佳抑制剂。

著录项

  • 作者

    Bareich, David C.;

  • 作者单位

    McMaster University (Canada).;

  • 授予单位 McMaster University (Canada).;
  • 学科 Biochemistry.;Microbiology.
  • 学位 Ph.D.
  • 年度 2003
  • 页码 181 p.
  • 总页数 181
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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