Hlavaty J J, Nowak T
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
Biochemistry. 1997 Mar 18;36(11):3389-403. doi: 10.1021/bi962255o.
Avian mitochondrial phosphoenolpyruvate carboxykinase (PEPCK) was incubated with Co2+ and H2O2 to form a stable Co3+-PEPCK complex. PEPCK, similarly incubated with H2O2 and either Mg2+ or Mn2+, resulted in no significant loss in activity over 30 min. PEPCK, incubated with Co2+ and H2O2 at pH 7.4, showed rapid inhibition as observed by a 40% decrease in activity after 5 min. The loss of activity is linear with the incorporation of cobalt into PEPCK, resulting in 15-25% activity for the stoichiometric Co3+-PEPCK complex. The incorporation of and inhibition by Co3+ is protected by PEP and GTP (ITP). Treatment of the Co3+-PEPCK complex with beta-mercaptoethanol results in a loss of cobalt and full recovery of activity. The reduction and reactivation are protected by PEP and GTP (ITP). EPR, PRR, circular dichroism, and fluorescence studies all indicate that Co3+ has been selectively incorporated into the cation site of PEPCK, resulting in a catalytically active enzyme-cation species. The substrates form Michaelis complexes with Co3+-PEPCK, and the catalytic reaction occurs as a second sphere complex as previously suggested [Lee & Nowak (1984) Biochemistry 23, 6506); Duffy & Nowak (1985) Biochemistry 24, 1152]. Proteolytic digestion of the Co3+-PEPCK complex and isolation of the cobalt-containing peptide by reverse phase HPLC were performed to identify the location of the cation binding site. From mass, amino acid composition, and sequence analyses of the isolated cobalt-peptide, the region Thr276-Lys301 is responsible for metal chelation. This very homologous region, located in the central portion of PEPCK, contains two highly conserved aspartic acids, Asp295 and Asp296, that are the only feasible metal binding ligands.
将禽源线粒体磷酸烯醇式丙酮酸羧激酶（PEPCK）与Co²⁺和H₂O₂一起孵育，以形成稳定的Co³⁺-PEPCK复合物。将PEPCK与H₂O₂以及Mg²⁺或Mn²⁺进行类似的孵育，在30分钟内活性没有显著损失。在pH 7.4条件下将PEPCK与Co²⁺和H₂O₂一起孵育，5分钟后活性下降40%，表现出快速抑制。活性损失与钴掺入PEPCK呈线性关系，化学计量的Co³⁺-PEPCK复合物的活性为15 - 25%。PEP和GTP（ITP）可保护Co³⁺的掺入和抑制作用。用β-巯基乙醇处理Co³⁺-PEPCK复合物会导致钴流失和活性完全恢复。PEP和GTP（ITP）可保护还原和再激活过程。电子顺磁共振（EPR）、脉冲弛豫速率（PRR）、圆二色性和荧光研究均表明，Co³⁺已被选择性地掺入PEPCK的阳离子位点，从而形成具有催化活性的酶-阳离子物种。底物与Co³⁺-PEPCK形成米氏复合物，催化反应如先前所述以第二球复合物的形式发生[Lee & Nowak（1984年）《生物化学》23卷，6506页；Duffy & Nowak（1985年）《生物化学》24卷，1152页]。对Co³⁺-PEPCK复合物进行蛋白酶消化，并通过反相高效液相色谱法分离含钴肽段，以确定阳离子结合位点的位置。通过对分离出的含钴肽段进行质谱、氨基酸组成和序列分析，发现Thr276 - Lys301区域负责金属螯合。这个高度同源的区域位于PEPCK的中部，包含两个高度保守的天冬氨酸，Asp295和Asp296，它们是唯一可行的金属结合配体。
