Oligopeptidases B (OpdB) belong to the family of serine prolyl oligopeptidases (clan SC, family S9) [1]. Like trypsin, OpdB hydrolyzes peptide bonds formed by the carbonyls of Arg/Lys residues, preferring Arg to Lys. Most preferable for hydrolysis residues in the P2 position of the substrate are also Arg or Lys [2]. The OpdB enzymes of ancient unicellular eukaryotes (Trypanosoma cruzi [3], T. brucei [4, 5], and T. evansi [6], as well as Leishmania major [5] and L. amazonensis [7]) are impor- tant virulent factors of these protozoan parasites. During trypanosomal infections (Chagas diseases, African sleep- ing sickness), OpdB is involved in the trypanosomal inva- sion of the cells [3] and abnormal degradation of peptide hormones of the host organism [6]. In mammals, genes encoding this enzyme have not been revealed. Thus, OpdB of protozoan parasites is a specific target for thera- peutic treatment of these dangerous infections. The OpdB enzyme and/or the gene encoding this enzyme have been also found in prokaryotes, namely in such Gram-negative pathogenic bacteria as Escherichia coli [8- 11], Moraxella lacunata [12], Salmonella enterica serovar typhimurium [13], mycobacteria Mycobacterium tubercu- losis and My. leprae [13], as well as in the spirochete Treponema denticola [14]. Bacterial OpdB proteins are much less studied; however, they also can be important targets for antimicrobial therapy [13]. In the psychrotolerant Gram-negative microorgan- ism S. proteamaculans 94 we revealed a novel trypsin-like proteinase (PSP) [15]. We obtained homogeneous (by PAGE) preparations of PSP (78 kDa), identified PSP as a previously unknown oligopeptidase B, sequenced the gene of OpdB S. proteamaculans 94, constructed a pro- ducer E. coli strain BL-21(DE3)/pOpdB, and developed a method for isolation of the recombinant enzyme His 6 - PSP (part I in this series [16]). The substrate specificity of PSP and the effect of calcium ions, pH, and temperature on the enzyme activity were investigated in detail (part II in this series [17]). It was demonstrated that the enzyme is psychrophilic, i.e. adapted to low temperatures [17]. Previously we demonstrated [15] that natural PSP was efficiently inhibited by inhibitors specific for serine proteinases (diisopropyl fluorophosphate, tosyl-lysine ISSN 0006-2979, Biochemistry (Moscow), 2012, Vol. 77, No. 3, pp. 300-306. © Pleiades Publishing, Ltd., 2012. Original Russian Text © A. G. Mikhailova, R. F. Khairullin, G. Ya. Kolomijtseva, L. D. Rumsh, 2012, published in Biokhimiya, 2012, Vol. 77, No. 3, pp. 384-391. 300 Abbreviations: AAS, atomic absorption spectroscopy; BAPNA, N α -benzoyl-DL-arginine-p-nitroanilide; BPTI, bovine basic pancreatic trypsin inhibitor; buffer A, 0.1 M Tris-HCl (pH 8.0); DMSO, dimethyl sulfoxide; IVA, inversion voltamperometry; OpdB, oligopeptidase B; PSP, proteinase from Serratia protea- maculans; p-NA, p-nitroanilide. * To whom correspondence should be addressed. Oligopeptidase B from Serratia proteamaculans. III. Inhibition Analysis. Specific Interactions with Metalloproteinase Inhibitors A. G. Mikhailova 1 *, R. F. Khairullin 1 , G. Ya. Kolomijtseva 2 , and L. D. Rumsh 1 1 Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia; fax: (495) 335-7103; E-mail: anna.mikhailova@ibch.ru 2 Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 939-3181; E-mail: kolom@genebee.msu.ru Received October 18, 2011 Revision received November 25, 2011 Abstract—Inhibition of the novel oligopeptidase B from Serratia proteamaculans (PSP) by basic pancreatic trypsin inhibitor, Zn 2+ ions, and o- and m-phenanthroline was investigated. A pronounced effect of calcium ions on the interaction of PSP with inhibitors was demonstrated. Inversion voltamperometry and atomic absorption spectrometry revealed no zinc ions in the PSP molecule. Hydrophobic nature of the enzyme inhibition by o- and m-phenanthroline was established. DOI: 10.1134/S0006297912030091 Key words: oligopeptidase B, Serratia proteamaculans, inhibition analysis, zinc-dependent enzymes, atomic absorption spectrometry, inversion voltamperometry