Phospholipase A2 engineering. Structural and functional roles of highly conserved active site residues tyrosine-52 and tyrosine-73.
Phospholipase A2 engineering. Structural and functional roles of highly conserved active site residues tyrosine-52 and tyrosine-73.
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磷脂酶A2工程。
DOI：
10.1021/bi00143a007
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Tsai,MD
中科院分区：
文献类型：
--
作者：
Dupureur,CM;Yu,BZ;Jain,MK;Noel,JP;Deng,T;Li,Y;Byeon,IJ;Tsai,MD
摘要：利用定点突变技术研究了两个高度保守的残基Tyr-52和Tyr-73在牛胰磷脂酶A2（PLA 2，在土杆菌中过量产生）界面催化中的结构和功能作用。根据晶体结构，这两个活性位点残基的侧链与催化残基Asp-99的羧酸根形成H-键。用Phe取代Tyr残基中的一个或两个，只导致催化速率的非常小的变化，这表明氢键对于PLA 2的催化作用不是必需的。用非芳族氨基酸取代Tyr残基导致在滑行模式动力学中朝向1，2-二辛酰基-s/i-甘油基-3-磷酸胆碱（DCgPC）胶束的表观和朝向1，2-二肉豆蔻酰基-sn-甘油基-3-磷酸甲醇（DC 14 PM）囊泡的v0（在最大底物浓度下的转换数，即摩尔分数= 1）的显著降低[贝格，O. G.，Yu，B.- Z.，罗杰斯，J.，& Jain，. K.（1991）Biochemistry 30，7283-7297]，进一步详细分析Y 52 V突变体的cooking模式动力学：通过荧光检测E至E* 平衡; E* S、E* P和E* I的解离常数（分别为Ks*、KP* 和Kf）通过组氨酸-48修饰的保护和差示紫外光谱法测量;在不存在和存在竞争性抑制剂的情况下，由水解的初始速率计算米氏常数Ku*;由v0和KM* 值计算饱和条件下的转换数（kat，这是理论值，因为酶在界面处可能不饱和）。结果表明，界面结合步骤（E至E*）中的扰动很小，但约。10-Ks*、K？*、*、Y 52 V功能的这种变化不是由于整体构象的变化，因为Y 52 V的质子NMR性质与野生型PLA 2的非常相似;相反，它可能是由活性位点处的酶-底物相互作用的扰动引起的。Tyr-73似乎发挥着重要的结构性作用。所有Tyr-73突变体的构象稳定性相对于野生型PLA 2的构象稳定性降低4-5 kcal/mol。质子核磁共振性质的Y 73 A建议显着的构象变化和大幅增加的构象灵活性。这些详细的结构和功能分析代表了在界面催化酶的结构-功能研究方面的一个重大进展。磷脂酶A2（PLA 2）1催化3-n-磷酸甘油酯的2-酰基酯键的水解。这种酶活性的核心是绝对保守的Asp-99和His-48，它们以丝氨酸蛋白酶的方式被称为催化二联体。与Asp-99氢键结合的是高度保守的Tyr-52和Tyr-73。除了蜂毒PLA 2（其仅具有一个类似的酪氨酸; Scott等人，
Revised Manuscript Received May 1, 1992 abstract: Site-directed mutagenesis was used toprobe the structural and functional roles of two highly conserved residues, Tyr-52 and Tyr-73, in interfacial catalysis by bovine pancreaticphospholipase A2 (PLA2, overproduced in Escherichiacoli). According to crystal structures, the side chains of these two active site residues form H-bonds with the carboxylate of the catalytic residue Asp-99. Replacement of either or both Tyr residues by Phe resulted in only very small changes in catalytic rates, which suggests that thehydrogen bonds are not essential for catalysis by PLA2. Substitution of either Tyr residue by nonaromatic amino acids resultedin substantial decreases in the apparent toward l, 2-dioctanoyl-s/i-glycero-3-phosphocholine (DCgPC) micelles and the v0 (turnover number at maximal substrate concentration, ie, mole fraction= 1) toward l, 2-dimyristoyl-sn-glycero-3-phosphomethanol (DC14PM) vesicles in scooting mode kinetics [Berg, O. G., Yu, B.-Z., Rogers, J., & Jain,. K.(1991) Biochemistry 30, 7283-7297], The Y52V mutant was further analyzed in detail byscooting mode kinetics: the E to E* equilibrium was examined by fluorescence; the dissociation constants of E* S, E* P, and E* I (Ks*, KP*, and Kf, respectively) in the presence of Ca2+ were measured by protection of histidine-48 modification and by difference UV spectroscopy; the Michaelis constant Ku* was calculated from initial rates of hydrolysis in the absence and presence of competitive inhibitors; and the turnover number under saturating conditions (kat, which is a theoretical value since the enzyme may not be saturated at the interface) was calculated from the v0 and KM* values. The results indicated little perturbation in the interfacial binding step (E to E*) but ca. 10-fold increases in Ks*, K?*,*, and KM* and a< 10-fold decrease in kaV Such changes in the function of Y52V are not due to global conformational changes since theproton NMR properties of Y52V closely resemble those of wild-type PLA2; instead, it is likely to be caused by perturbed enzyme-substrate interactions at the active site. Tyr-73 appears to play an important structural role. The conformational stability of allTyr-73 mutants decreased by 4-5 kcal/mol relative to that of the wild-type PLA2. The proton NMR properties of Y73A suggested significant conformational changes and substantially increased conformational flexibility. These detailedstructural and functional analyses represent a major advancement in the structure-function study of an enzyme involved in interfacial catalysis.Riospholipase A2 (PLA2) 1 catalyzes the hydrolysis of the 2-acyl ester bond of 3-jn-phosphoglycerides. Central to the activity of this enzyme are the absolutely conserved Asp-99 and His-48, termed the catalytic dyad in the fashion of serine proteases. Hydrogen-bonded to Asp-99 are the highly con-served Tyr-52 and Tyr-73. With the exception of bee venom PLA2 (which has only one analogous tyrosine; Scott et al.,
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磷脂相A2的结构和功能
影响因子：
--
作者：
H. Verheij;A. J. Slotboom;G. Haas
通讯作者：
G. Haas
磷脂酶 A2：结构主题的变化及其对机制的影响
DOI：
--
发表时间：
1987
期刊：
影响因子：
--
作者：
J. Maraganore;R. Poorman;R. Heinrikson
通讯作者：
R. Heinrikson
DOI：
10.1111/j.1432-1033.1983.tb07859.x
发表时间：
1983-01-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
DUFTON, MJ;EAKER, D;HIDER, RC
通讯作者：
HIDER, RC
影响因子：
2.9
作者：
BAUM, J;DOBSON, CM;HANLEY, C
通讯作者：
HANLEY, C
亚胶束浓度的带负电荷的去垢剂诱导猪胰磷脂酶 A2 及其酶原的聚集。
影响因子：
2.9
作者：
J. Hille;M. Egmond;R. Dijkman;M. V. van Oort;B. Jirgensons;G. de Haas
通讯作者：
G. de Haas