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Effect of reactive center loop structure of antichymotrypsin on inhibition of duodenase activity

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Abstract

Interaction between duodenase (a granase family member) from bovine duodenal mucosa and recombinant antichymotrypsin (rACT) and its P1 variants has been studied. Association rate constants (k a) were 11, 6.8, and 17 mM−1·sec−1 for rACT, ACT L358M, and ACT L358R, respectively. Natural antitrypsin (AT) compared to ACT was a 20 times more effective duodenase inhibitor (in terms of k a). Duodenase interacted with P1 variants of ACT via a suicide mechanism with stoichiometry of the process SI = 1.2. The nature of the P1 residue of the inhibitor did not influence the interaction if other residues did not meet conformational requirements of the duodenase substrate-binding pocket. Also, interaction of duodenase with ACT variants containing residues from AT reaction center loop (rACT P2-P3′, rACT P3-P4′, rACT P4-P3′, and rACT P6-P4′) was studied. The inhibition type ([E]0 = 1·10−7 M, 25°C) was revealed to be reversible-like, and efficacy of inhibition decreased with increase in the substituted part of the reactive center loop. Constants of inhibition (K i) were measured. Efficacy of interaction between the enzyme (duodenase) and inhibitor depends on topochemical correspondence between a substrate-binding pocket of the enzyme and substrate structure.

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Abbreviations

(r)ACT:

(recombinant) antichymotrypsin

AT:

α1-antitrypsin

BBI:

soybean Bowman-Birk inhibitor

BTEE:

N-benzoyl-L-tyrosine ethyl ester

CT:

chymotrypsin

RCL:

reaction center loop

SI:

stoichiometry of inhibition

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Authors and Affiliations

  1. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia

    T. S. Zamolodchikova

  2. Faculty of Chemistry, Lomonosov Moscow State University, 119992, Moscow, Russia

    N. A. Popykina, I. P. Gladysheva & N. I. Larionova

  3. Cardiovascular Research Laboratory, Department of Medicine, Medical College of Georgia, Augusta, GA, 30912, USA

    I. P. Gladysheva

Authors
  1. T. S. Zamolodchikova
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  2. N. A. Popykina
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  3. I. P. Gladysheva
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  4. N. I. Larionova
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Corresponding authors

Correspondence to T. S. Zamolodchikova or N. A. Popykina.

Additional information

Original Russian Text © T. S. Zamolodchikova, N. A. Popykina, I. P. Gladysheva, N. I. Larionova, 2009, published in Biokhimiya, 2009, Vol. 74, No. 8, pp. 1015–1027.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM08-286, March 1, 2009.

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Zamolodchikova, T.S., Popykina, N.A., Gladysheva, I.P. et al. Effect of reactive center loop structure of antichymotrypsin on inhibition of duodenase activity. Biochemistry Moscow 74, 824–833 (2009). https://doi.org/10.1134/S0006297909080021

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  • DOI: https://doi.org/10.1134/S0006297909080021

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