Biochemistry (Moscow)

, Volume 83, Issue 12–13, pp 1594–1602 | Cite as

Mobile Loop in the Active Site of Metallocarboxypeptidases as an Underestimated Determinant of Substrate Specificity

  • V. Kh. AkparovEmail author
  • V. I. Timofeev
  • I. G. Khaliullin
  • E. G. Konstantinova
  • I. P. Kuranova
  • T. V. Rakitina
  • V. K. Švedas


It is generally accepted that the primary specificity of metallocarboxypeptidases is mainly determined by the structure of the so–called primary specificity pocket. However, the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT) with the primary specificity pocket fully reproducing the one in pancreatic carboxypeptidase B (CPB) retained the broad, mainly hydrophobic substrate specificity of the wild–type enzyme. In order to elucidate factors affecting substrate specificity of metallocarboxypeptidases and the reasons for the discrepancy with the established views, we have solved the structure of the complex of the CPT G215S/A251G/T257A/D260G/T262D mutant with the transition state analogue N–sulfamoyl–L–phenylalanine at a resolution of 1.35 Å and compared it with the structure of similar complex formed by CPB. The comparative study revealed a previously underestimated structural determinant of the substrate specificity of metallocarboxypeptidases and showed that even if substitution of five amino acid residues in the primary specificity pocket results in its almost complete structural correspondence to the analogous pocket in CPB, this does not lead to fundamental changes in the substrate specificity of the mutant enzyme due to the differences in the structure of the mobile loop located at the active site entrance that affects the substrate–induced conformational rearrangements of the active site.


metallocarboxypeptidase T from Thermoactinomyces vulgaris metallocarboxypeptidase B S1′–subsite substrate selectivity N–sulfamoyl–L–phenylalanine X–ray analysis 



buried surface area


carboxypeptidase B from porcine pancreas


carboxypeptidase T from Thermoactinomyces vulgaris


CPT mutant with A243G, T250A, A253G, D260G, T262D substitutions


wild–type carboxypeptidase T


carboxypeptidase U


carboxypeptidase O






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Copyright information

© Pleiades Publishing, Inc. 2018

Authors and Affiliations

  • V. Kh. Akparov
    • 1
    Email author
  • V. I. Timofeev
    • 2
    • 3
  • I. G. Khaliullin
    • 4
  • E. G. Konstantinova
    • 1
  • I. P. Kuranova
    • 2
    • 3
  • T. V. Rakitina
    • 3
    • 5
  • V. K. Švedas
    • 6
  1. 1.State Research Institute for Genetics and Selection of Industrial MicroorganismsMoscowRussia
  2. 2.Shubnikov Institute of Crystallography, Crystallography and Photonics Federal Scientific Research CenterRussian Academy of SciencesMoscowRussia
  3. 3.Kurchatov Institute National Research CenterMoscowRussia
  4. 4.Laboratory of Ion and Molecular PhysicsMoscow Institute of Physics and Technology (State University)Dolgoprudny, Moscow RegionRussia
  5. 5.Shemyakin−Ovchinnikov Institute of Bioorganic Chemistry, Laboratory of Hormonal Regulation ProteinsRussian Academy of SciencesMoscowRussia
  6. 6.Belozersky Institute of Physico-Chemical BiologyLomonosov Moscow State UniversityMoscowRussia

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