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

V. Kh. Akparov1,a*, V. I. Timofeev2,3,b, I. G. Khaliullin4,c, G. E. Konstantinova1, I. P. Kuranova2,3,b, T. V. Rakitina3,5,d, and V. K. Švedas6,e

1State Research Institute for Genetics and Selection of Industrial Microorganisms, 117545 Moscow, Russia

2Shubnikov Institute of Crystallography, Crystallography and Photonics Federal Scientific Research Center, Russian Academy of Sciences, 119333 Moscow, Russia

3Kurchatov Institute National Research Center, 123098 Moscow, Russia

4Laboratory of Ion and Molecular Physics, Moscow Institute of Physics and Technology (State University), 141700 Dolgoprudny, Moscow Region, Russia

5Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Laboratory of Hormonal Regulation Proteins, Russian Academy of Sciences, 117997 Moscow, Russia

6Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

* To whom correspondence should be addressed.

Received June 5, 2018; Revised September 19, 2018; Accepted September 19, 2018
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.
KEY WORDS: metallocarboxypeptidase T from Thermoactinomyces vulgaris, metallocarboxypeptidase B, S1′-subsite, substrate selectivity, N-sulfamoyl-L-phenylalanine, X-ray analysis

DOI: 10.1134/S0006297918120167