Role of tyrosine 129 in the active site of spinach glycolate oxidase.
Role of tyrosine 129 in the active site of spinach glycolate oxidase.
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DOI：
10.1111/j.1432-1033.1993.tb17852.x
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发表时间：
1993-05
期刊：
影响因子：
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通讯作者：
P. Macheroux;V. Kieweg;V. Massey;E. Söderlind;K. Stenberg;Y. Lindqvist
中科院分区：
文献类型：
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作者：
P. Macheroux;V. Kieweg;V. Massey;E. Söderlind;K. Stenberg;Y. Lindqvist
研究了菠菜乙醇酸氧化酶（Y129 F乙醇酸氧化酶）的酶学性质和三维结构。突变体的结构是未扰动的，这有利于解释生化数据。Y129 F乙醇酸氧化酶的吸收光谱在364和450 nm处具有最大值（λ max = 11400 M-1 cm-1）。光谱表明黄素处于其正常质子化形式，即Y129 F突变体不像野生型酶那样降低氧化黄素的N（3）的pKa [Macheroux，P.，Massey，V.，Thiele，D. J.，和Volokita，M.（1991）Biochemistry 30，4612-4619]。这通过Y129 F乙醇酸氧化酶的pH滴定证实，其显示pKa高于pH 9。与野生型乙醇酸氧化酶相反，草酸盐不会干扰Y129 F乙醇酸氧化酶的吸收光谱。此外，草酸盐不抑制突变酶的酶活性。野生型乙醇酸氧化酶的典型特征与黄素N（1）-C（2 = O）附近带正电荷的赖氨酸侧链相关，例如阴离子黄素半醌的稳定化和紧密N（5）-亚硫酸盐加合物的形成，这些特征在Y129 F突变蛋白中都是保守的。Y129 F乙醇酸氧化酶表现出约3.5%的野生型活性。与野生型酶的20 s-1相比，突变体的较低转换数为0.74 s-1，相当于过渡态能量增加约7.8 kJ/mol。稳态分析得出乙醇酸和氧气的Km值分别为1.5 mM和7 μ M。乙醇酸的Km略高于野生型乙醇酸氧化酶（1 mM），而氧的Km低得多。与野生型乙醇酸氧化酶的情况一样，还原被发现是催化中的限速步骤，速率为0.63 s-1。Y129 F乙醇酸氧化酶的动力学性质提供了证据，表明Tyr 129的羟基的主要功能是稳定过渡态。
The enzymatic properties and the three-dimensional structure of spinach glycolate oxidase which has the active-site Tyr129 replaced by Phe (Y129F glycolate oxidase) has been studied. The structure of the mutant is unperturbed which facilitates interpretation of the biochemical data. Y129F glycolate oxidase has an absorbance spectrum with maxima at 364 and 450 nm (epsilon max = 11400 M-1 cm-1). The spectrum indicates that the flavin is in its normal protonated form, i.e. the Y129F mutant does not lower the pKa of the N(3) of oxidized flavin as does the wild-type enzyme [Macheroux, P., Massey, V., Thiele, D. J., and Volokita, M. (1991) Biochemistry 30, 4612-4619]. This was confirmed by a pH titration of Y129F glycolate oxidase which showed that the pKa is above pH 9. In contrast to wild-type glycolate oxidase, oxalate does not perturb the absorbance spectrum of Y129F glycolate oxidase. Moreover oxalate does not inhibit the enzymatic activity of the mutant enzyme. Typical features of wild-type glycolate oxidase that are related to a positively charged lysine side chain near the flavin N(1)-C(2 = O), such as stabilization of the anionic flavin semiquinone and formation of tight N(5)-sulfite adducts, are all conserved in the Y129F mutant protein. Y129F glycolate oxidase exhibited about 3.5% of the wild-type activity. The lower turnover number for the mutant of 0.74 s-1 versus 20 s-1 for the wild-type enzyme amounts to an increase of the energy of the transition state of about 7.8 kJ/mol. Steady-state analysis gave Km values of 1.5 mM and 7 microM for glycolate and oxygen, respectively. The Km for glycolate is slightly higher than that found for wild-type glycolate oxidase (1 mM) whereas the Km for oxygen is much lower. As was the case for wild-type glycolate oxidase, reduction was found to be the rate-limiting step in catalysis, with a rate of 0.63 s-1. The kinetic properties of Y129F glycolate oxidase provide evidence that the main function of the hydroxyl group of Tyr129 is the stabilization of the transition state.