Effect of Reactive Center Loop Structure of Antichymotrypsin on Inhibition of Duodenase Activity

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T. S. Zamolodchikova^1*, N. A. Popykina², I. P. Gladysheva^2,3, and N. I. Larionova²

¹Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia; E-mail: tatyana@enzyme.siobc.ras.ru

²Faculty of Chemistry, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-5417; E-mail: natpopykina@mail.ru

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

^* To whom correspondence should be addressed.

Received September 8, 2008; Revision received November 17, 2008

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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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KEY WORDS: duodenase, serpins, conformational specificity, granase

DOI: 10.1134/S0006297909080021

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