Kinetic properties of native and mutagenized isoforms of mitochondrial alcohol dehydrogenase III purified from Kluyveromyces lactis
Kinetic properties of native and mutagenized isoforms of mitochondrial alcohol dehydrogenase III purified from Kluyveromyces lactis
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DOI：
10.1016/j.biochi.2004.08.004
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发表时间：
2004-09-01
期刊：
影响因子：
3.9
通讯作者：
Bozzi, A
中科院分区：
文献类型：
--
作者：
Brisdelli, F;Saliola, M;Bozzi, A
通过计算机建模和蛋白质工程，我们研究了位于酵母乳酸克鲁维酵母线粒体醇脱氢酶III的辅酶口袋中的两个氨基酸残基的变化。这两个残基Gly 225和Ala 274被假设参与NAD（H）和NADP（H）之间的酶区分。在将Gly 225改变为Ala后，我们产生了与野生型差异很小的酶（突变体G225 A）。与此相反，在274位改变Phe而不是Ala（MuA 274 F）导致NAD（P）和NADPH的Ki值显著增加，甚至催化活性更显著降低。在该突变体中，NADP（H）的k（cat）/K-m率也降低。在225和274处具有双重变化的酶（突变体G225 A-A274 F）显示，除了NADPH的显著低K值和其高催化效率之外，相对于辅酶的动力学参数在单取代上不是加和的。令人惊讶的是，在两个位置处具有变化的酶有效地减少乙醛，相对于亲本或单一突变的酶，显示出低10倍的Ki值和高7倍的催化效率。没有一种工程酶会转化甲醛、戊二醛或芳香醛，但所有酶都以乙醛所表现出的相对反应速率的大约一半还原丙醛和丁醛。有趣的是，只有突变体A274 F能够氧化甲醇几乎以及乙醇。此外，该突变体能够转换仲醇和环状醇，在其他异构体中未检测到的速率。这些结果与预测大体一致，即增加辅酶口袋附近的氨基酸的大小将阻碍NADP的调节，但与NADPH以及具有高空间位阻的醇或乙醇底物的亲和力增加不一致。(C)2004年，Elsevier SAS。All rights reserved.
By computer modelling and protein engineering we have investigated changes in two amino acid residues located in the coenzyme pocket of the yeast Kluyveromyces lactis mitochondrial alcohol dehydrogenase III. These two residues, Gly 225 and Ala 274, were hypothesized to be involved in the enzyme discrimination between NAD(H) and NADP(H). Upon changing Gly 225 to Ala we produced an enzyme (mutant G225A) showing very little difference from the wild-type. On the contrary, change at position 274 of Phe instead of Ala (mutantA274F) caused a significant increase of K, values for NAD(P) and for NADPH and even a more marked decrease in catalytic activity. The k(cat)/K-m rates for NADP(H) were also decreased in this mutant. Enzymes with the double changes at 225 and 274 (mutant G225A-A274F) showed, apart the substantial low K, value for NADPH and its high catalytic efficiency, kinetic parameters relative to coenzymes which were not additive over the single substitutions. Surprisingly, enzymes with changes at the two positions reduced efficiently acetaldehyde, displaying a K, value 10-fold lower and a catalytic efficiency sevenfold higher with respect to parent or singularly mutated enzymes. None of the engineered enzymes would convert formaldehyde, glutaraldehyde or aromatic aldehydes but all enzymes reduced propionaldehyde and butyraldehyde at relative reaction rates approximately half of that exhibited by acetaldehyde. Interestingly only mutant A274F was able to oxidize methanol almost as well as ethanol. In addition, this mutant was capable to convert secondary and cyclic alcohols, at a rate not detected in the other isoforms. These results are in general agreement with the prediction that increasing the size of amino acids in the proximity of the coenzyme pocket would hamper the accommodation of NADP but discord the increased affinity for NADPH as well as for alcoholic or aldehydic substrates with high steric hindrance. (C) 2004 Elsevier SAS. All rights reserved.