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首页> 外文期刊>Biochemistry >Galactose mutarotase: Purification, characterization, and investigations of two important histidine residues
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Galactose mutarotase: Purification, characterization, and investigations of two important histidine residues

机译：半乳糖诱变酶：两个重要组氨酸残基的纯化，表征和研究

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Galactose mutarotase catalyzes the interconversion of alpha- and beta-anomers of aldoses and is a recently identified member of the gal operon of Escherichia coli and participant in the Leloir pathway [Bouffard et al. (1994) J. Mol. Biol. 244, 269-278]. We report the purification and characterization of this enzyme, as well as mechanistic studies involving chemical modification with diethylpyrocarbonate (DEPC) and site-directed mutagenesis demonstrating the significance of two conserved histidine residues. The enzyme lacks metal ions and oxidoreduction cofactors, and an extinction coefficient of (6.2 +/- 0.4) x 10(4) M-1 cm(-1) has been measured by quantitative amino acid analysis. The catalytic mechanism is likely concerted general acid/general base. Experiments involving modification with DEPC suggest that a histidine is essential and is protected by substrate. Furthermore, site-directed mutagenesis of two conserved histidines was performed, and characterization of these mutants (His104Gln and His175Asn) illustrates the significance of these residues. Kinetic analysis of H104Q demonstrates an increase in K-M of about 600-fold, a decrease in k(cat) of approximately 7-fold, and a 4000-fold decrease in kcat/K-M as compared to the wild-type enzyme. The activity of His175Asn mutant, on the other hand, was too low to be measured accurately, and His 175 remains a candidate for the general base. These mutants were also subjected to DEPC modification, and results are consistent with the presence of two important histidines positioned closely together in the active site. [References: 39]
机译：半乳糖诱变酶催化醛糖的α-和β-端基异构体的相互转化，并且是最近鉴定的大肠杆菌gal操纵子的成员，并且是Leloir途径的参与者[Bouffard等人。 （1994）J.Mol。生物学244，269-278]。我们报告了这种酶的纯化和表征，以及涉及用焦碳酸二乙酯（DEPC）进行化学修饰和定点诱变的机理研究，证明了两个保守的组氨酸残基的重要性。该酶缺乏金属离子和氧化还原辅助因子，消光系数为（6.2 +/- 0.4）x 10（4）M-1 cm（-1），已通过定量氨基酸分析测定。催化机理可能是协调一致的通用酸/通用碱。涉及用DEPC修饰的实验表明，组氨酸是必不可少的，并受底物保护。此外，进行了两个保守的组氨酸的定点诱变，这些突变体（His104Gln和His175Asn）的表征说明了这些残基的重要性。 H104Q的动力学分析表明，与野生型酶相比，K-M升高约600倍，k（cat）降低约7倍，kcat / K-M降低4000倍。另一方面，His175Asn突变体的活性太低，无法准确测量，而His 175仍然是一般碱基的候选者。这些突变体也经过了DEPC修饰，其结果与两个重要的组氨酸在活性位点中紧密排列的存在一致。 [参考：39]

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