Mechanism of the reaction catalyzed by delta 5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: kinetic properties of a modified enzyme in which tyrosine 14 is replaced by 3-fluorotyrosine.
Mechanism of the reaction catalyzed by delta 5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: kinetic properties of a modified enzyme in which tyrosine 14 is replaced by 3-fluorotyrosine.
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睾丸酮丛毛单胞菌 (假单胞菌) δ 5-3-酮类固醇异构酶催化的反应机制：其中酪氨酸 14 被 3-氟酪氨酸取代的修饰酶的动力学特性。
DOI：
10.1021/bi00175a042
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Benisek,WF
中科院分区：
文献类型：
--
作者：
Brooks,B;Benisek,WF
A5-3-酮甾体异构酶的酪氨酸14在该酶的功能中起重要作用，因为它被苯丙氨酸取代导致Eca减少1CH· 7倍。这一结果以及该残基存在于酶的底物结合位点并且与结合的类固醇的C-2非常接近的事实表明，它在催化机制中通过底物的C-3羰基氧的质子化或氢键结合而起到亲电体的作用。为了获得更多关于酪氨酸14作用的信息，我们制备了一种修饰形式的酶，其中酪氨酸14在体内基本上被外源提供的3-氟酪氨酸取代，3-氟酪氨酸是一种酪氨酸衍生物，其中酚羟基的pKf应降低约1.5 log单位。通过将非必需酪氨酸55和88的密码子突变为苯丙氨酸，确保了这种修饰的位点特异性。我们发现Y55中的酪氨酸14被3-氟酪氨酸取代。88 F修饰的异构酶导致kCit 4倍的降低。我们解释了这一结果的机制，烯醇化的过渡态是二烯醇样的，其特征是相对较少的质子转移从酪氨酸14在过渡态，和中间体在整个反应是二烯醇样的。来自睾丸酮丛毛单胞菌（以前称为假单胞菌）的Δ 5 -3-酮甾体异构酶（KSI）1（Tamaoka等人，1987）催化底物的4/3质子转移到产物的6/3位置。伴随着这种转移的是底物的5，6双键迁移到
Revised Manuscript Received October 25, 1993® abstract: Tyrosine 14 of A5-3-ketosteroid isomerase plays an important role in the function of the enzyme, since itsreplacement by phenylalanine results in a decrease in£ ca, by a factor of 1CH· 7. This resultand the fact that this residue resides in the enzyme’s substrate binding site and is in close proximity to C-2 of the bound steroid suggests that it functions as an electrophile in the catalytic mechanism by protonation of or hydrogen bonding to the C-3 carbonyl oxygen of the substrate. In order to obtain more information about the role of tyrosine 14, we have prepared a modified form of the enzyme in which tyrosine 14 has been substantially replaced in vivo by exogenously supplied 3-fluorotyrosine, a tyrosine derivative in which the pKf of the phenol hydroxyl should be decreased by about 1.5 log units. Site specificity of this modification has been ensuredby mutation of the codons for the nonessential tyrosines 55 and 88 to phenylalanine. We find that replacement of tyrosine 14 by 3-fluorotyrosine in the Y55. 88F modifiedform of the isomerase results in a 4-fold decrease in kCit-We interpret this result in terms of a mechanism in which the transition state for enolization is dienolate-like, characterized by relatively little proton transfer from tyrosine 14 in the transition state, and the intermediate in the overall reaction is dienol-like. An alternative mechanism in which the intermediate is stabilized by a short, strong hydrogen bond can also be consistent with the data.The A5-3-ketosteroid isomerase (KSI) 1 from Comamonas (formerly Pseudomonas) testosteroni (Tamaoka et al., 1987) catalyzes the transfer of the 4/3 proton of the substrate to the 6/3 position in the product. Concomitant with this transfer is the migration of the 5, 6 double bond of the substrate to the