Department of Biochemistry, The University of Iowa, Iowa City, IA 52242-1109, USA.
Chem Biol Interact. 2013 Feb 25;202(1-3):104-10. doi: 10.1016/j.cbi.2012.11.010. Epub 2012 Nov 27.
Previous studies showed that fitter yeast (Saccharomyces cerevisiae) that can grow by fermenting glucose in the presence of allyl alcohol, which is oxidized by alcohol dehydrogenase I (ADH1) to toxic acrolein, had mutations in the ADH1 gene that led to decreased ADH activity. These yeast may grow more slowly due to slower reduction of acetaldehyde and a higher NADH/NAD(+) ratio, which should decrease the oxidation of allyl alcohol. We determined steady-state kinetic constants for three yeast ADHs with new site-directed substitutions and examined the correlation between catalytic efficiency and growth on selective media of yeast expressing six different ADHs. The H15R substitution (a test for electrostatic effects) is on the surface of ADH and has small effects on the kinetics. The H44R substitution (affecting interactions with the coenzyme pyrophosphate) was previously shown to decrease affinity for coenzymes 2-4-fold and turnover numbers (V/Et) by 4-6-fold. The W82R substitution is distant from the active site, but decreases turnover numbers by 5-6-fold, perhaps by effects on protein dynamics. The E67Q substitution near the catalytic zinc was shown previously to increase the Michaelis constant for acetaldehyde and to decrease turnover for ethanol oxidation. The W54R substitution, in the substrate binding site, increases kinetic constants (Ks, by >10-fold) while decreasing turnover numbers by 2-7-fold. Growth of yeast expressing the different ADHs on YPD plates (yeast extract, peptone and dextrose) plus antimycin to require fermentation, was positively correlated with the log of catalytic efficiency for the sequential bi reaction (V1/KiaKb=KeqV2/KpKiq, varying over 4 orders of magnitude, adjusted for different levels of ADH expression) in the order: WT≈H15R>H44R>W82R>E67Q>W54R. Growth on YPD plus 10mM allyl alcohol was inversely correlated with catalytic efficiency. The fitter yeast are "bradytrophs" (slow growing) because the ADHs have decreased catalytic efficiency.
先前的研究表明，能够在有丙烯醇存在的情况下通过发酵葡萄糖生长的酵母（酿酒酵母）更加适应环境，这些酵母中的醇脱氢酶 I（ADH1）基因发生了突变，导致 ADH 活性降低。由于乙醛的还原速度较慢，NADH/NAD(+) 比值较高，丙烯醇的氧化速度应该会降低，因此这些酵母的生长速度可能会较慢。我们确定了三种具有新定点取代的酵母 ADH 的稳态动力学常数，并研究了在选择性培养基上表达六种不同 ADH 的酵母的生长与催化效率之间的相关性。H15R 取代（用于测试静电效应）位于 ADH 的表面，对动力学的影响很小。H44R 取代（影响与辅酶焦磷酸的相互作用）先前已被证明会使辅酶的亲和力降低 2-4 倍，周转率（V/Et）降低 4-6 倍。W82R 取代远离活性部位，但会使周转率降低 5-6 倍，可能是通过对蛋白质动力学的影响。先前已经证明，E67Q 取代靠近催化锌的位置会增加乙醛的米氏常数，并降低乙醇氧化的周转率。W54R 取代位于底物结合位点，会增加动力学常数（Ks，增加 10 倍以上），同时使周转率降低 2-7 倍。在 YPD 平板（酵母提取物、蛋白胨和葡萄糖）上表达不同 ADH 的酵母的生长，加上需要发酵的抗霉素，与顺序生物反应的催化效率的对数呈正相关（V1/KiaKb=KeqV2/KpKiq，变化幅度超过 4 个数量级，根据不同的 ADH 表达水平进行调整），顺序为：WT≈H15R>H44R>W82R>E67Q>W54R。在 YPD 加 10mM 丙烯醇上的生长与催化效率呈负相关。适应性更强的酵母是“缓慢生长菌”（生长缓慢），因为 ADH 的催化效率降低。
