Stereochemical control of yeast reductions. 5. Characterization of the oxidoreductases involved in the reduction of .beta.-keto esters
Stereochemical control of yeast reductions. 5. Characterization of the oxidoreductases involved in the reduction of .beta.-keto esters
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酵母还原的立体化学控制。
DOI：
10.1021/ja00296a038
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发表时间：
1985
影响因子：
15
通讯作者：
C. Sih
中科院分区：
文献类型：
--
作者：
W. Shieh;A. Gopalan;C. Sih
R= C2H5 1.82 303 1.00 0.21 1.00 6.60 R= C4H9 1.33 202 0.10 0.11 0.094 7.13 R= c6h，R= C8 H17 1.60 69 0.29 0.47 0.01 6.12面包酵母通过改变酯取代基的大小。2在本文中，我们研究了支配这一惊人的立体化学观察的潜在因素。我们现在报告的分离三个占主导地位的竞争/3-酮氧化还原酶，并显示酯取代基与特异性常数，3，4 kCM/K的明显相关性。此外，乙基（7 R）-现在可以通过使用缺少相反立体化学偏好的竞争酶之一的酿酒酵母突变体来制备高光学纯度（ee= 0.90）的4-氯-3-羟基丁酸酯（5）。我们从面包酵母（Red星星）的胞质级分中纯化了三种酶，它们能有效地还原4-氯乙酰乙酸酯，产生相应的高光学纯度的甲醇产物（> 0.97 ee）。它们都优先利用NADPH作为辅酶。这些酶（MW 2 400 000）具有的物理和化学性质，让人联想到脂肪酸合成酶6和减少β-酮酯，产生D构型的甲醇。7虽然其他两种酶的天然底物尚未确定，但它们可以在羟基磷灰石柱上容易地分离。较快移动的蛋白质（d酶）具有38000的MW，并且还原β-酮酯以产生d甲醇产物，而l酶（MW 74000）提供l构型的甲醇。由于哺乳动物L-3-（羟酰基）-CoA还原酶8（EC 1：1：35）在NADH存在下将4-氯乙酰乙酸酯还原为高对映体过量（> 0.97）的（R）-4-氯-3-羟基丁酸酯，我们仔细地在面包酵母中寻找这种还原酶活性。虽然面包酵母的线粒体部分积极减少乙酰乙酰辅酶A，只有微量的还原酶活性是可检测的使用1或3作为底物。这一观察结果表明，不同物种的L-3-（羟酰基）-CoA脱氢酶在底物特异性方面具有显著差异。9为了深入了解酯取代基对完整面包酵母对7-氯-/3-酮酯的对映选择性还原的影响，测量了三种酶对各种4-氯乙酰乙酸酯的kQM（转换数）和K（米氏常数）（表1）。注意到随着酯基团的扩大，脂肪酸合成酶的kaJK 4适度降低（图1）。辛酯的kM/K的更显著的下降可归因于Iceat值的降低（表I）。当L酶的酯基从乙基变为辛基时，kc l/K值的增加更为显著（图1）。有趣的是，这种增加是由于K的减少（表I）。这些com-
R= C2H5 1.82 303 1.00 0.21 1.00 6.60 R= C4H9 1.33 202 0.10 0.11 0.094 7.13 R= c6h, 3 1.82 252 0.20 0.23 0.028 6.87 R= C8H17 1.60 69 0.29 0.47 0.01 6.12 bakers’ yeast via modification of the size of the ester substituent. 2 In thispaper, we examine the underlying factors governing this striking stereochemical observation. We now report the isolation of three dominant competing/3-keto oxidoreductases and show a distinct correlation of the ester substituent with the specificity constant, 3, 4 kCM/K. Moreover, ethyl (7?)-4-chloro-3-hydroxybutanoate (5) of high optical purity (ee= 0.90) may now be prepared by using a mutant of S', cerevisiae lacking one of the competing enzymes of opposite stereochemical preference. We have purified three enzymes to homogeneity from the cytosolic fraction of bakers’ yeast (Red Star) capable of actively reducing 4-chloroacetoacetic esters to yield corresponding car-binolic products of high optical purity5 (> 0.97 ee). All of them utilize NADPH preferentially as the coenzyme. One of these enzymes (MW 2 400 000) possesses physical and chemical properties reminiscent of fatty acid synthetase6 and reduces/3-keto esters to yield carbinols of D configuration. 7 Although the natural substrates for the other two enzymes have not yet been defined they may be readily resolved on a hydroxyapatite column. The faster moving protein (d enzyme) has an MW of 38 000 and reduces/3-keto esters to yield d carbinolic products, whereas l enzyme (MW 74000) affords carbinols of l configuration. Be-cause mammalian L-3-(hydroxyacyl)-CoA dehydrogenase8 (EC 1: 1: 35) in the presence of NADH reduced 4-chloroacetoacetic esters to (R)-4-chloro-3-hydroxybutanoates of high enantiomeric excess (> 0.97), we carefully searched for this reductase activity in bakers’ yeast. While mitochondrial fractions of bakers’ yeast actively reduced acetoacetyl-CoA, only a trace of reductase activity was detectable using either 1 or 3 as substrates. This observation indicates that L-3-(hydroxyacyl)-CoA dehydrogenases of different species have marked differences in substrate specificities. 9 To gain an insight into the influence of the ester substituent on the enantioselective reduction of 7-chloro-/3-keto esters by intact bakers’ yeast, the kQM (turnover number) and K (Michaelis con-stant) for the three enzymes on various 4-chloroacetoacetic esters were measured (Table I).A moderate decrease in kaJK4 is noted forfatty acid synthetase as the ester grouping is enlarged (Figure 1). The more pronounced drop in kM/K for the octyl ester is attributable to a decrease in the value of/ceat (Table I). More dramatic is the increase in the value of kc¡¡ l/K as the ester grouping is changed from ethyl to octyl for the L enzyme (Figure 1). Interestingly, this increase is due to a decrease in K (Table I). These com-