MECHANISTIC STUDIES OF THE FLAVOPROTEIN TRYPTOPHAN 2-MONOOXYGENASE .2. PH AND KINETIC ISOTOPE EFFECTS
MECHANISTIC STUDIES OF THE FLAVOPROTEIN TRYPTOPHAN 2-MONOOXYGENASE .2. PH AND KINETIC ISOTOPE EFFECTS
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DOI：
10.1021/bi00011a029
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发表时间：
1995-03-21
期刊：
影响因子：
2.9
通讯作者：
FITZPATRICK, PF
中科院分区：
文献类型：
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作者：
EMANUELE, JJ;FITZPATRICK, PF
以色氨酸、苯丙氨酸、2-肼-3-丙酸和蛋氨酸为底物，测定了pH值和动力学同位素对黄蛋白色氨酸2-单加氧酶稳态动力学参数的影响。氨基酸底物的V/K值表明，表观pK(a)值为5的残基必须未质子化才能产生活性，pK(a)值等于底物氨基的残基必须被质子化，pK(a)值为10的残基去质子化会增加V/K值。游离酶中pK(A)值为6的基团为了与酰胺抑制剂紧密结合必须去质子化，为了与酸紧密结合必须去质子化，从而将其作为固有的pK值。该pK(a)值的温度依赖性与组氨酸残基的参与是一致的。pK(a)值为10的残基去质子化降低了酰胺抑制剂的结合。在pH 5 ~ 10之间，(D)（V/K-trp）值小于1.7，符合该底物对7 ~ 15的正向催化作用。(D)(V/K)（ala）值与pH值有关，从pH 8.3时的最小值1.8增加到高、低pH时的极限值5.3,pK(a)值分别为5.1和10。高pH下同位素效应和V/K-met值的增加与pH 10以上构象向更开放的活性位点转变相一致。pH 8.3时(D)(V/K)（ala）值为5.3；这可能是这个底物的本征同位素效应。[β, β, β - h -2(3)]丙氨酸的β -次级同位素效应为0.965 +/- 0.041，符合碳离子机制。pK(a)值为6的残基的作用是去除底物α -质子，形成碳离子。苯丙氨酸和色氨酸的V/K-O2值在pH 5和pH 10之间基本不敏感。添加蛋氨酸后，V/K-O2值增加数倍，pK(a)值为6.8；这是分配给减少的FAD。与色氨酸反应的V/K-O2值没有溶剂同位素效应，与氧反应的限速电子转移一致。对于所有三种底物，当单个残基质子化时，V-max值降低20-50倍。该pK(a)值随基质的不同而变化；它被分配到产品发布之前的构象变化。溶剂同位素对色氨酸V-max值的影响为2.5。这与缓慢的质子转移与产物释放相结合是一致的。
pH and kinetic isotope effects on steady-state kinetic parameters have been determined for the flavoprotein tryptophan 2-monooxygenase with tryptophan, phenylalanine, 2-hydrazino-3-propanoic acid, and methionine as substrates. The V/K values of the amino acid substrates show that a residue with an apparent pK(a) value of 5 must be unprotonated for activity, a residue with a pK(a) value equal to that of the amino group of the substrate must be protonated, and deprotonation of a residue with pK(a) value of 10 increases the V/K value. A group in the free enzyme with a pK(a) value of 6 must be deprotonated for tight binding of amide inhibitors and protonated for tight binding of acids, establishing this as the intrinsic pK, value. The temperature dependence of this pK(a) value is consistent with involvement of a histidinyl residue. Deprotonation of the residue with a pK(a) value of 10 decreases binding of amide inhibitors. The (D)(V/K-trp) value is less than 1.7 between pH 5 and 10, consistent with a forward commitment to catalysis of 7-15 with this substrate. The (D)(V/K)(ala) value is pH dependent, increasing from a minimal value of 1.8 at pH 8.3 to a limiting value of 5.3 at both high and low pH, with pK(a) values of 5.1 and 10. The increase in both the isotope effect and the V/K-met value at high pH is consistent with a conformational change to a more open active site above pH 10. The (D)(V/K)(ala) value is 5.3 at pH 8.3; this is probably the intrinsic isotope effect with this substrate. The beta-secondary isotope effect with [beta,beta,beta-H-2(3)]alanine is 0.965 +/- 0.041, consistent with a carbanion mechanism. The proposed role of the residue with a pK(a) value of 6 is to remove the substrate alpha-proton to form the carbanion. The V/K-O2 values for phenylalanine and tryptophan are essentially insensitive to pH between pH 5 and pH 10. The V/K-O2 value with methionine increases severalfold with a pK(a) value of 6.8; this is assigned to the reduced FAD. There is no solvent isotope effect on the V/K-O2 value with tryptophan, consistent with rate-limiting electron transfer in the reaction with oxygen. With all three substrates, the V-max value decreases 20-50 fold when a single residue is protonated. This pK(a) value varies with the identity of the substrate; it is assigned to a conformational change which precedes product release. The solvent isotope effect on the V-max value with tryptophan is 2.5. This is consistent with slow proton transfer being coupled to product release.