Peters G H, Frimurer T M, Olsen O H
MedChem Research IV, Novo-Nordisk A/S, Mâlov, Denmark.
Biochemistry. 1998 Apr 21;37(16):5383-93. doi: 10.1021/bi971187i.
The catalytic activity of protein-tyrosine phosphatases (PTPs) is mediated by a cysteine side chain which carries out a nucleophilic attack initiating the phosphate cleavage. Experimentally, it has been observed that the active site cysteine has a remarkably low pKa. In the present study, we have investigated the origin of the low pKa by analyzing the electrostatic properties of four different protein-tyrosine phosphatases: Yersinia PTP (bacteria), PTP1B (human), VHR (human), and low molecular weight phosphatase (bovine). These phosphatases have very low sequence homology and show very low structural similarity. However, they share a common active site motif [the (H/V)CX5R(S/T) sequence] which adopts a unique loop structure. We have applied the so-called single site titration method, which is based on the Poisson-Boltzmann methodology, to (i) study the influence of the architecture of the (H/V)CX5R(S/T) loop on the pKa of the active cysteine and (ii) examine which parts of the active site region stabilize the ionized form of the cysteine. Our results indicate that the architecture of the (H/V)CX5R(S/T) loop has a major impact on the low pKa of the active cysteines. The orientation of the microdipoles generated by the partial charges of the backbone atoms (i.e., the CONHCalpha atoms) is essential for maintaining the low pKa. Further, the electrostatic field generated by these microdipoles has a larger impact than the electrostatic dipole generated by the central alpha-helix. Interactions of the active cysteine with other ionizable side chains play a minor role in stabilizing the thiolate anion. The only ionizable side chain significantly influencing the pKa of the active site cysteine is the arginine, which is an important part of the consensus sequence.
蛋白质酪氨酸磷酸酶（PTPs）的催化活性由一个半胱氨酸侧链介导，该侧链进行亲核攻击以启动磷酸酯裂解。实验观察到，活性位点半胱氨酸的pKa值极低。在本研究中，我们通过分析四种不同的蛋白质酪氨酸磷酸酶的静电性质来探究低pKa值的来源，这四种酶分别是：耶尔森氏菌PTP（细菌）、PTP1B（人类）、VHR（人类）和低分子量磷酸酶（牛）。这些磷酸酶的序列同源性非常低，结构相似性也很低。然而，它们共享一个共同的活性位点基序[（H/V）CX5R（S/T）序列]，该基序采用独特的环结构。我们应用了基于泊松-玻尔兹曼方法的所谓单位点滴定法，来（i）研究（H/V）CX5R（S/T）环的结构对活性半胱氨酸pKa值的影响，以及（ii）检查活性位点区域的哪些部分稳定了半胱氨酸的离子化形式。我们的结果表明，（H/V）CX5R（S/T）环的结构对活性半胱氨酸的低pKa值有重大影响。由主链原子（即CONHCalpha原子）的部分电荷产生的微偶极子的取向对于维持低pKa值至关重要。此外，这些微偶极子产生的静电场比中央α-螺旋产生的静电偶极子的影响更大。活性半胱氨酸与其他可电离侧链的相互作用在稳定硫醇阴离子方面起次要作用。唯一对活性位点半胱氨酸的pKa值有显著影响的可电离侧链是精氨酸，它是共有序列的重要组成部分。
