The structural basis for the perturbed pKa of the catalytic base in 4-oxalocrotonate tautomerase:: Kinetic and structural effects of mutations of Phe-50
The structural basis for the perturbed pKa of the catalytic base in 4-oxalocrotonate tautomerase:: Kinetic and structural effects of mutations of Phe-50
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DOI：
10.1021/bi0024714
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发表时间：
2001-02-20
期刊：
影响因子：
2.9
通讯作者：
Whitman, CP
中科院分区：
文献类型：
--
作者：
Czerwinski, RM;Harris, TK;Whitman, CP
4-草草酸互变异构体酶（4-OT）的氨基端脯氨酸由于其异常低的pK(a)为6.4 +/- 0.2，比模型化合物脯氨酸酰胺低3个单位，因此在酶催化β， γ -不饱和烯酮向α， β -异构体异构化的过程中起着一般碱催化剂的作用。最近的研究表明，这种异常低的pK(a)不是由于附近的阳离子残基（Arg-11， Arg-39和Arg-61）的静电效应[Czerwinski， R. M., Harris， T. K., Johnson, Jr.， W. H., Legler， P. M., Stivers， J. T., Mildvan, a . S., Whitman， C. P.（1999）生物化学38,12358-1.2366]。因此，它可能仅仅是由于在疏水活性部位的低局部介电常数为14.7 +/- 0.8。对活性位点ph -50突变体的研究支持了这一机制，该突变体距离Pro-1为5.8埃，是Pro-1 9埃范围内的12个极性残基之一。用Tyr替代phe50不会显著改变k（cat）或k -m，并且通过N-15核磁共振光谱测定Pro-1的pK(a)为6.0 +/- 0.1，与野生型相当。F50Y突变体的H-1-N-15 HSQC和3D H-1-N-15 NOESY HSQC光谱显示其构象与野生型酶非常相似。在F50Y突变体中，Tyr-50的pK(a)比模型化合物n -乙酰酪氨酸酰胺的pK(a)增加了两个单位，达到12.2 +/- 0.3，通过W和H-1 NMR滴定测定，产生了13.4 +/- 1.7的局部介电常数，与该突变体中Pro-i的降低的pK(a)测定的13.7 +/- 0.3值一致。在F50A突变体中，通过N-15核磁共振滴定，Pro-1的pK(a)为7.3 +/- 0.1，与pH / k(cat)/ k -m速率谱中的pK(a)为7.6 +/- 0.2相当，比野生型酶的pK(a)大一个单位，表明局部介电常数增加到21.2 +/- 2.6。在野生型4-OT中发现的F50A突变体中4个链间NOE和1个转NOE的消失表明，从残基50到57的β -发夹（覆盖活性位点，也是突变位点）的结构丢失。F50A突变体的H-1-N-15 HSQC光谱显示主链N-15和NH的化学位移发生了广泛而大的变化，包括Gly残基48、51、53和54的化学位移，导致它们在23°c时失去分散，在43°c时由于与溶剂的快速交换而消失。这些观察结果证实，F50A突变体的活性位点更容易进入外部水环境，导致Pro-1的局部介电常数和pK(a)增加。此外，与野生型酶相比，F50A突变使k（cat）降低了167倍，k -m增加了11倍，这表明除了降低Pro-1的pK(a)外，疏水环境在催化过程中也起着重要作用。F50I和F50V突变使蛋白不稳定，k（cat）分别降低58倍和1.6倍，k -m分别增加3.3倍和3.8倍。
The amino-terminal proline of 4-oxalocrotonate tautomerase (4-OT) functions as the general base catalyst in the enzyme-catalyzed isomerization of beta,gamma -unsaturated enones to their alpha,beta -isomers because of its unusually low pK(a) of 6.4 +/- 0.2, which is 3 units lower than that of the model compound, proline amide. Recent studies show that this abnormally low pK(a) is not due to the electrostatic effects of nearby cationic residues (Arg-11, Arg-39, and Arg-61) [Czerwinski, R. M., Harris, T. K., Johnson, Jr., W. H., Legler, P. M., Stivers, J. T., Mildvan, A. S., and Whitman, C. P. (1999) Biochemistry 38, 12358-1.2366]. Hence, it may result solely from a low local dielectric constant of 14.7 +/- 0.8 at the otherwise hydrophobic active site. Support for this mechanism comes from the study of mutants of the active site Phe-50, which is 5.8 Angstrom from Pro-1 and is one of 12 apolar residues within 9 Angstrom of Pro-1. Replacing Phe-50 with Tyr does not significantly alter k(cat) or K-m and results in a pK(a) of 6.0 +/- 0.1 for Pro-1 as determined by N-15 NMR spectroscopy, comparable to that observed for wild type. H-1-N-15 HSQC and 3D H-1-N-15 NOESY HSQC spectra of the F50Y mutant demonstrate its conformation to be very similar to that of the wild-type enzyme. In the F50Y mutant, the pK(a) of Tyr-50 is increased by two units from that of a model compound N-acetyl-tyrosine amide to 12.2 +/- 0.3, as determined by W and H-1 NMR titrations, yielding a local dielectric constant of 13.4 +/- 1.7, in agreement with the value of 13.7 +/- 0.3 determined from the decreased pK(a) of Pro-i in this mutant. In the F50A mutant, the pK(a) of Pro-1 is 7.3 +/- 0.1 by N-15 NMR titration, comparable to the pK(a) of 7.6 +/- 0.2 found in the pH vs k(cat)/K-m rate profile, and is one unit greater than that of the wild-type enzyme, indicating an increase in the local dielectric constant to a value of 21.2 +/- 2.6. A loss of structure of the beta -hairpin from residues 50 to 57, which covers the active site, and is the site of the mutation, is indicated by the disappearance in the F50A mutant of four interstrand NOEs and one turn NOE found in wild-type 4-OT. H-1-N-15 HSQC spectra of the F50A mutant reveal widespread and large changes in the backbone N-15 and NH chemical shifts including those of Gly residues 48, 51, 53, and 54 causing their loss of dispersion at 23 degreesC and their disappearance at 43 degreesC due to rapid exchange with solvent. These observations confirm that the active site of the F50A mutant is more accessible to the external aqueous environment, causing an increase in the local dielectric constant and in the pK(a) of Pro-1. In addition, the F50A mutation decreased k(cat) 167-fold and increased K-m 11-fold from those of the wildtype enzyme, suggesting an important role for the hydrophobic environment in catalysis, beyond that of decreasing the pK(a) of Pro-1. The F50I and F50V mutations destabilize the protein and decrease k(cat) by factors of 58 and 1.6, and increase K-m by 3.3- and 3.8-fold, respectively.