Mobile Loop in the Active Site of Metallocarboxypeptidases as an Underestimated Determinant of Substrate Specificity
- Authors: Akparov V.K.1, Timofeev V.I.2,3, Khaliullin I.G.4, Konstantinova E.G.1, Kuranova I.P.2,3, Rakitina T.V.3,5, Švedas V.K.6
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Affiliations:
- State Research Institute for Genetics and Selection of Industrial Microorganisms
- Shubnikov Institute of Crystallography, Crystallography and Photonics Federal Scientific Research Center
- Kurchatov Institute National Research Center
- Laboratory of Ion and Molecular Physics
- Shemyakin−Ovchinnikov Institute of Bioorganic Chemistry, Laboratory of Hormonal Regulation Proteins
- Belozersky Institute of Physico-Chemical Biology
- Issue: Vol 83, No 12-13 (2018)
- Pages: 1594-1602
- Section: Article
- URL: https://journals.rcsi.science/0006-2979/article/view/151789
- DOI: https://doi.org/10.1134/S0006297918120167
- ID: 151789
Cite item
Abstract
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.
About the authors
V. Kh. Akparov
State Research Institute for Genetics and Selection of Industrial Microorganisms
Author for correspondence.
Email: valery.akparov@yandex.ru
Russian Federation, Moscow, 117545
V. I. Timofeev
Shubnikov Institute of Crystallography, Crystallography and Photonics Federal Scientific Research Center; Kurchatov Institute National Research Center
Email: valery.akparov@yandex.ru
Russian Federation, Moscow, 119333; Moscow, 123098
I. G. Khaliullin
Laboratory of Ion and Molecular Physics
Email: valery.akparov@yandex.ru
Russian Federation, Dolgoprudny, Moscow Region, 141700
E. G. Konstantinova
State Research Institute for Genetics and Selection of Industrial Microorganisms
Email: valery.akparov@yandex.ru
Russian Federation, Moscow, 117545
I. P. Kuranova
Shubnikov Institute of Crystallography, Crystallography and Photonics Federal Scientific Research Center; Kurchatov Institute National Research Center
Email: valery.akparov@yandex.ru
Russian Federation, Moscow, 119333; Moscow, 123098
T. V. Rakitina
Kurchatov Institute National Research Center; Shemyakin−Ovchinnikov Institute of Bioorganic Chemistry, Laboratory of Hormonal Regulation Proteins
Email: valery.akparov@yandex.ru
Russian Federation, Moscow, 123098; Moscow, 117997
V. K. Švedas
Belozersky Institute of Physico-Chemical Biology
Email: valery.akparov@yandex.ru
Russian Federation, Moscow, 119991