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

T. S. Zamolodchikova1*, N. A. Popykina2, I. P. Gladysheva2,3, 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
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 (ka) 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 ka). 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 (Ki) 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.
KEY WORDS: duodenase, serpins, conformational specificity, granase

DOI: 10.1134/S0006297909080021