Studies on the reaction mechanism of Rhodotorula gracilis D-amino-acid oxidase -: Role of the highly conserved Tyr-223 on substrate binding and catalysis
Studies on the reaction mechanism of Rhodotorula gracilis D-amino-acid oxidase -: Role of the highly conserved Tyr-223 on substrate binding and catalysis
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DOI：
10.1074/jbc.274.51.36233
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发表时间：
1999-12-17
影响因子：
4.8
通讯作者：
Pollegioni, L
中科院分区：
文献类型：
--
作者：
Harris, CM;Molla, G;Pollegioni, L
我们用定点突变的方法对细长红酵母的D-氨基酸氧化酶进行了研究，以确定是否有可能在酸碱催化中起作用的残基。D-氨基酸氧化酶中为数不多的保守残基之一Tyr-223已经突变为苯丙氨酸和丝氨酸，这两个突变体都是D-丙氨酸转化的活性催化剂，它们被D-丙氨酸还原的速度略快于野生型酶。Tyr-223->；Phe突变体与野生型酶几乎完全相同，而Tyr-223->；Ser突变体的底物结合速度比野生型慢60倍，产物释放速度至少慢800倍。这些数据排除了Tyr-223作为活性中心酸碱催化剂的可能性。这些结果强调了Tyr-223对底物结合的重要性，并例证了空间相互作用在RgDAAO催化中的重要性。
We have studied D-amino-acid oxidase from Rhodotorula gracilis by site-directed mutagenesis for the purpose of determining the presence or absence of residues having a possible role in acid/base catalysis, Tyr-223, one of the very few conserved residues among D-amino-acid oxidases, has been mutated to phenylalanine and to serine, Both mutants are active catalysts in turnover with D-alanine, and they are reduced by D-alanine slightly faster than wild type enzyme. The Tyr-223 -> Phe mutant is virtually identical to the wild-type enzyme, whereas the Tyr-223 -> Ser mutant exhibits 60-fold slower substrate binding and at least 800-fold slower rate of product release relative to wild-type. These data eliminate Tyr-223 as an active site acid/base catalyst. These results underline the importance of Tyr-223 for substrate binding and exemplify the importance of steric interactions in RgDAAO catalysis.