Taton M, Husselstein T, Benveniste P, Rahier A
Département d'Enzymologie Moléculaire et Cellulaire, Institut de Biologie Moléculaire des Plantes, CNRS UPR 406, 28 rue Goethe, 67083, Strasbourg Cédex, France.
Biochemistry. 2000 Feb 1;39(4):701-11. doi: 10.1021/bi991467t.
The role of 15 residues in the reaction catalyzed by Arabidopsis thaliana Delta7-sterol-C5(6)-desaturase (5-DES) was investigated using site-directed mutagenesis and expression of the mutated enzymes in an erg3 yeast strain defective in 5-DES. The mutated desaturases were assayed in vivo by sterol analysis and quantification of Delta5,7-sterols. In addition, the activities of the recombinant 5-DESs were examined directly in vitro in the corresponding yeast microsomal preparations. One group of mutants was affected in the eight evolutionarily conserved histidine residues from three histidine-rich motifs. Replacement of these residues by leucine or glutamic acid completely eliminated the desaturase activity both in vivo and in vitro, in contrast to mutations at seven other conserved residues. Thus, mutants H203L, H222L, H222E, P201A, G234A, and G234D had a 5-DES activity reduced to 2-20% of the wild-type enzyme, while mutants K115L, P175V, and P175A had a 5-DES activity and catalytical efficiency (V/K) that was similar to that of the wild-type. Therefore, these residues are not essential for the catalysis but contribute to the activity through conformational or other effects. One possible function for the histidine-rich motifs would be to provide the ligands for a presumed catalytic Fe center, as previously proposed for a number of integral membrane enzymes catalyzing desaturations and hydroxylations [Shanklin et al. (1994) Biochemistry 33, 12787-12794]. Another group of mutants was affected in residue 114 based on previous in vivo observations in A. thaliana indicating that mutant T114I was deficient in 5-DES activity. We show that the enzyme T114I has an 8-fold higher Km and 10-fold reduced catalytic efficiency. Conversely, the functionally conservative substituted mutant enzyme T114S displays a 28-fold higher Vmax value and an 8-fold higher Km value than the wild-type enzyme. Consequently, V/K for T114S was 38-fold higher than that for T114I. The data suggest that Thr 114 is involved in stabilization of the enzyme-substrate complex with a marked discrimination between the ground-state and the transition state of a rate-controlling step in the catalysis by the 5-DES.
利用定点诱变技术，并在缺乏5-去饱和酶（5-DES）的erg3酵母菌株中表达突变酶，研究了15个残基在拟南芥Delta7-甾醇-C5(6)-去饱和酶（5-DES）催化反应中的作用。通过甾醇分析和Delta5,7-甾醇的定量分析，对体内突变的去饱和酶进行了测定。此外，还在相应的酵母微粒体制剂中直接对重组5-DES的活性进行了体外检测。一组突变体中，来自三个富含组氨酸基序的八个进化保守组氨酸残基受到影响。用亮氨酸或谷氨酸取代这些残基，会完全消除体内和体外的去饱和酶活性，这与其他七个保守残基的突变情况形成对比。因此，突变体H203L、H222L、H222E、P201A、G234A和G234D的5-DES活性降至野生型酶的2%-20%，而突变体K115L、P175V和P175A的5-DES活性和催化效率（V/K）与野生型相似。因此，这些残基对催化作用并非必不可少，但通过构象或其他效应有助于活性的发挥。富含组氨酸基序的一个可能功能是为推测的催化铁中心提供配体，正如之前对许多催化去饱和和羟基化的整合膜酶所提出的那样[尚克林等人（1994年），《生物化学》33卷，12787-12794页]。另一组突变体基于之前在拟南芥中的体内观察结果，发现114位残基受到影响，表明突变体T114I缺乏5-DES活性。我们发现，T114I酶的米氏常数（Km）高8倍，催化效率降低10倍。相反，功能保守取代的突变体酶T114S的最大反应速度（Vmax）值比野生型酶高28倍，Km值高8倍。因此，T114S的V/K比T114I高38倍。数据表明，苏氨酸114参与了酶-底物复合物的稳定，在5-DES催化的速率控制步骤的基态和过渡态之间存在明显差异。
