PROBING THE PRESUMED CATALYTIC TRIAD OF SELENIUM-CONTAINING PEROXIDASES BY MUTATIONAL ANALYSIS OF PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE-PEROXIDASE (PHGPX)
PROBING THE PRESUMED CATALYTIC TRIAD OF SELENIUM-CONTAINING PEROXIDASES BY MUTATIONAL ANALYSIS OF PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE-PEROXIDASE (PHGPX)
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DOI：
10.1515/bchm3.1995.376.11.651
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发表时间：
1995-11-01
期刊：
影响因子：
--
通讯作者：
FLOHE, L
中科院分区：
文献类型：
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作者：
MAIORINO, M;AUMANN, KD;FLOHE, L
对影响硒酶PHGPx假定催化中心的单、双位突变体进行了功能分析，估算了基态酶氧化和再生的速率常数k(+1)和k‘(+2)，并分析了反应中心被碘乙酸酯烷基化的速率(k(Inact))，半胱氨酸(PHGPx(Cys46))取代具有催化活性的硒半胱氨酸46使k(+1)和k’(+2)减少了约三个数量级，但不影响k(Inact)。此外，涉及三联体其他残基的PHGPx(Cys46)突变同时减少了k(Inact)和k(+1)，从而突出了Gln 81和Trp 136参与了亲核半胱氨酸硫醇的解离/激活。总体而言，PHGPx(Cys46)中的酸性残基取代Gin 81或Trp 136最显著地降低了k(+1)值，因为它们实际上阻止了硫醇基团的解离，而这些位置上的中性或带正电的残基允许和中间解离，并诱导了相应的硫醇反应活性。我们的数据首次揭示了硒半胱氨酸、谷氨酰胺和色氨酸残基的三者代表了一种新型的催化中心，其完整性对于谷胱甘肽过氧化物酶的全部催化功能是必不可少的。
Single and double site mutants affecting the presumed catalytic centre of the selenoenzyme PHGPx were subjected to functional analysis, The rate constants k(+1) and k'(+2), for the oxidation and the regeneration of the ground state enzyme were estimated, respectively, Moreover, the alkylation rate of the reactive centre by iodoacetate (k(inact)) was also analysed, The substitution of the catalytically competent selenocysteine 46 by cysteine (PHGPx(cys46)) decreased k(+1) and k'(+2) by about three orders of magnitude, although leaving unaffected k(inact). Furthermore, mutations of PHGPx(cys46) involving the other residues of the triad decreased both k(inact), and k(+1), thus highlighting the involvement of Gln 81 and Trp 136 in the dissociation/activation of the nucleophilic cysteine thiol. In general, substitutions of Gin 81 or Trp 136 by acidic residues in PHGPx(cys46) most dramatically depressed the k(+1) values, because they practically prevented the dissociation of the thiol group, while neutral or positively charged residues in these positions allowed and intermediate dissociation and induced a corresponding reactivity of the thiol. Our data, for the first time, reveal that the presumed triad of selenocysteine, glutamine and tryptophan residues represents a novel type of catalytic centre, whose integrity is essential for the full catalytic function of glutathione peroxidases.