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首页> 外文期刊>Biochemistry >Structure and dynamics of the metal site of cadmium-substituted carboxypeptidase A in solution and crystalline states and under steady-state peptide hydrolysis
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Structure and dynamics of the metal site of cadmium-substituted carboxypeptidase A in solution and crystalline states and under steady-state peptide hydrolysis

机译：镉取代的羧肽酶A在溶液，结晶态和稳态肽水解下金属位点的结构和动力学

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摘要

PAC spectra (perturbed angular correlation of gamma-rays) of cadmium-substituted carboxypeptidase A (CPD) show that the enzyme in solution imposes a flexible, pH- and chloride-dependent coordination structure on the metal site, in contrast to what is found in the crystalline state. A much more restricted coordination geometry occurs for the steady-state peptide intermediates of Bz-Gly-l-Phe and Bz-Gly-Gly-l-Phe in solution, suggesting that substrate binding locks the structure in a rigid conformation. The results further indicate that the peptide intermediate has a six-coordinated metal coordination geometry with an OH- ligand at the solvent site and a carbonyl oxygen at an additional ligand site. In marked contrast, conformational rigidity is not induced by the inhibitor/poor substrate Gly-L-Tyr nor by the products of high turnover substrates, Bz-Gly, Bz-Gly-Gly, and L-Phe. These results are consistent with an intact scissile peptide bond in the enzyme-substrate complex of Bz-Gly-L-Phe and Bz-Gly-Gly-L-Phe. A single nuclear quadrupole interaction (NQI) is observed for the crystalline state of the enzyme between pH 5.7 and pH 9.4. This NQI agrees with calculations based on the metal coordination geometry for cadmium in crystalline CPD derived from X-ray diffraction studies. A single broad distribution of NQIs is observed for CPD in sucrose solutions and 0.1 M NaCl at pH values below 6.5. This NQI (NQI-1') has parameters very close to those for the crystalline state. The enzyme metal site, characterized by this NQI, is converted into two new enzyme metal sites over the pH range of 6.5-8.3. The metal coordination sphere of one of these has a NQI (NQI-1) with parameters similar to those at lower pH values (NQI-1') while the other NQI (NQI-2) is characterized by markedly different NQI parameters. Angular overlap model (AOM) calculations indicate that the coordination sites giving NQI-1' and NQI-1 both have a metal-bound water molecule while the coordination site giving NQI-2 has a metal-bound hydroxide ion. PAC results at pH 8.3-10.5 indicate that in this pH range the two metal coordination geometries related to NQI-1 and NQI-2 occur in a pH independent ratio of 2:1, with the one with the water ligand being the most abundant species. The observed pH-independent equilibrium between the two different metal coordination geometries for cadmium can be explained by an equilibrium between tautomeric forms of a hydrogen bond between the Glu-270 carboxyl group and the metal-bound water (Glu-270 COO-...(HOH)M <==> Glu-270 COOH...(OH-)M) being slow on the time scale of a PAC experiment, i.e., slower than 0.5 micros. We finally suggest that NQI-1' observed at low pH reflects an enzyme species containing a metal-coordinated water molecule and the protonated carboxyl group of Glu-270.
机译：镉取代的羧肽酶A（CPD）的PAC光谱（伽马射线的扰动角相关性）显示，与在金属中发现的相反，溶液中的酶在金属位点上施加了一种灵活的，pH和氯化物依赖性的配位结构。结晶状态。溶液中Bz-Gly-1-Phe和Bz-Gly-Gly-1-Phe的稳态肽中间体发生的配位几何形状受到更多限制，这表明底物结合将结构锁定为刚性构象。结果还表明，该肽中间体具有六配位的金属配位几何结构，在溶剂位点具有OH-配体，在另一个配体位点具有羰基氧。形成鲜明对比的是，抑制剂/不良底物Gly-L-Tyr或高周转底物Bz-Gly，Bz-Gly-Gly和L-Phe的产物均不诱导构象刚性。这些结果与Bz-Gly-L-Phe和Bz-Gly-Gly-L-Phe的酶-底物复合物中的完整易断裂肽键一致。在pH 5.7和pH 9.4之间观察到酶的结晶状态的单核四极相互作用（NQI）。该NQI与基于X射线衍射研究得出的结晶CPD中镉的金属配位几何结构的计算结果相符。在低于6.5的pH值下，在蔗糖溶液和0.1 M NaCl中观察到CPD的NQI分布很宽。该NQI（NQI-1'）的参数非常接近晶态。以NQI为特征的酶金属位点在6.5-8.3的pH范围内被转化为两个新的酶金属位点。其中一个的金属配位球具有一个NQI（NQI-1），其参数与较低pH值（NQI-1'）相似，而另一个NQI（NQI-2）则具有明显不同的NQI参数。角重叠模型（AOM）计算表明，给出NQI-1'和NQI-1的配位点都具有与金属结合的水分子，给出NQI-2的配位点具有与金属结合的氢氧离子。 PAC在pH值8.3-10.5下的结果表明，在此pH值范围内，与NQI-1和NQI-2相关的两种金属配位几何结构的pH无关比例为2：1，其中水配体含量最高的一种。镉在两种不同的金属配位几何结构之间观察到的与pH无关的平衡可以通过Glu-270羧基和与金属结合的水（Glu-270 COO -...）之间的氢键互变异构形式之间的平衡来解释。 （HOH）M Glu-270 COOH ...（OH-）M）在PAC实验的时间尺度上较慢，即，慢于0.5微米。我们最后建议在低pH下观察到的NQI-1'反映了一种酶物种，该酶物种包含金属配位的水分子和Glu-270的质子化羧基。

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