X- ray diffraction analysis revealed the role of the L254 residue in the recognition of the substrate by carboxypeptidase T from Thermoactinomyces vulgaris

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Crystal structures of complexes of the mutant protein L254N carboxypeptidase T from Thermoactinomyces vulgaris with stable transition state analogues -N- sulfamoyl-L-glutamate, N-sulfamoyl-L-arginine, N-sulfamoyl-L-valine and N-sulfamoyl-L-leucine (resolution 2.05, 1.89, 2.30, 1.79 Å) were obtained. The dependence of the association constants of these inhibitors, as well as the efficiency of catalysis of the corresponding tripeptide substrates ZAAX, on the distances between the atoms of the ligand O15, O16, O20, T19 and the active center of the mutant protein N146, Y225 and E277 was found. This dependence differs significantly from the previously identified dependence for wild-type carboxypeptidase T. The results obtained indicate the involvement of leucine 254, which is part of the mobile loop of metallocarboxypeptidases, in the discrimination of substrates by carboxypeptidase T according to the induced fit mechanism.

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V. Akparov

National Research Center “Kurchatov Institute”

编辑信件的主要联系方式.
Email: valery.akparov@yandex.ru

Shubnikov Institute of Crystallography of the Kurchatov Complex Crystallography and Photonics

俄罗斯联邦, Moscow

V. Timofeev

National Research Center “Kurchatov Institute”

Email: valery.akparov@yandex.ru

Shubnikov Institute of Crystallography of the Kurchatov Complex Crystallography and Photonics

俄罗斯联邦, Moscow

G. Konstantinova

National Research Center “Kurchatov Institute”

Email: valery.akparov@yandex.ru

Shubnikov Institute of Crystallography of the Kurchatov Complex Crystallography and Photonics

俄罗斯联邦, Moscow

I. Kuranova

National Research Center “Kurchatov Institute”

Email: valery.akparov@yandex.ru

Shubnikov Institute of Crystallography of the Kurchatov Complex Crystallography and Photonics

俄罗斯联邦, Moscow

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2. Fig. 1. Correlation between the –lgKi values for the interaction of N-sulfamoyl-X transition state analogs and kcat/Km for the hydrolysis of the corresponding tripeptide substrates (ZAAX, where X is a variable amino acid residue) using the wild-type CPT (diamonds) and the mutant CPT protein L254N (squares).

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3. Fig. 2. The location of N-sulfamoyl inhibitors SArg (a), SGlu (b), SLeu (c), SVal (d) in the active site of CPTwt/CPTL254N. The mutant protein is highlighted in light.

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4. Fig. 3. Dependence of the association constants of sulfamoyl inhibitors with KPTwt and the mutant protein KPT L254N on the bond length between O15 of the sulfamoyl group and ND2 of ASN146 (a), and O16 and Y255[OH] (b). Both oxygen atoms of the ligand mimic the oxygen atoms of the C-terminal carboxyl of the substrate. Squares are the values related to the mutant protein L254N.

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5. Fig. 4. Dependences of the logarithms of the association constants (a, c) of sulfamoyl inhibitors with KPTwt and KPT L254N and the efficiency of catalysis (b) on the distances between the inhibitor atoms (N19, S, O20) and the atoms of the residues of the catalytic centers N19-E277[OE1] (a), O20-E277[OE2] (b), S-Zn2+ (c), included in the tetrahedral transition complex. Data for KPTwt are diamonds, for KPT L254N – squares.

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