Role of tyrosine 114 of L-methionine y-lyase from Pseudomonas putida
Role of tyrosine 114 of L-methionine y-lyase from Pseudomonas putida
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DOI：
10.1271/bbb.64.2336
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发表时间：
2000-11-01
影响因子：
1.6
通讯作者：
Tanaka, H
中科院分区：
文献类型：
--
作者：
Inoue, H;Inagaki, K;Tanaka, H
恶臭假单胞菌的l -蛋氨酸γ -裂解酶在活性位点有一个保守的酪氨酸残基（Tyr114），在所有已知的γ -家族吡哆醛5'-磷酸依赖酶的序列中也是如此。在l -蛋氨酸γ -裂解酶的突变体中，ty114被苯丙氨酸（Y114F）取代，导致l -蛋氨酸α、γ -消除的k（cat）降低了910倍，而k -m与野生型酶相同。在Y114F突变体中，对于在γ -位置有一个吸电子基团的底物，即o -乙酰- l-高丝氨酸和l-蛋氨酸砜，k（cat）分别降低了28倍和16倍，对于α、β -消除和脱胺底物，k（cat）也有类似的降低。突变酶催化的底物交换氢反应和底物-酶复合物的光谱变化表明，在Y114F突变体的α、γ消除总反应中，l -蛋氨酸的γ消除过程是限速的决定步骤。这些结果表明，l -蛋氨酸γ -裂解酶的Tyr114在消除底物中起重要作用。
L-Methionine gamma -lyase from Pseudomonas putida has a conserved tyrosine residue (Tyr114) in the active site as in all known sequences of gamma -family pyridoxal 5'-phosphate dependent enzymes. A mutant form of L-methionine gamma -lyase in which Tyr114 was replaced by phenylalanine (Y114F) resulted in 910-fold decrease in k(cat) for alpha,gamma -elimination of L-methionine, while the K-m remained the same as the wild type enzyme. The Y114F mutant had the reduced k(cat) by only 28- and 16-fold for substrates with an electron-withdrawing group at the gamma -position, namely O-acetyl-L-homoserine and L-methionine sulfone, respectively, and also the similar reduction of k(cat) for alpha,beta -elimination and deamination substrates. The hydrogen exchange reactions of substrate and the spectral changes of the substrate-enzyme complex catalyzed by the mutant enzyme suggested that gamma -elimination process for L-methionine is the rate-limiting determination step in alpha,gamma -elimination overall reaction of the Y114F mutant. These results indicate that Tyr114 of L-methionine gamma -lyase is important in gamma -elimination of the substrate.