Oligopeptidase B from Serratia proteamaculans. II. Enzymatic Characteristics: Substrate Analysis, Influence of Calcium Ions, pH and Temperature Dependences

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A. G. Mikhailova^1*, R. F. Khairullin¹, I. V. Demidyuk², T. Yu. Gromova², S. V. Kostrov², and L. D. Rumsh¹

¹Shemyakin 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

²Institute of Molecular Genetics, Russian Academy of Sciences, pl. Kurchatova 2, 123182 Moscow, Russia; fax: (495) 196-0221; E-mail: duk@img.ras.ru

^* To whom correspondence should be addressed.

Received November 2, 2010; Revision received November 18, 2010

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Enzymatic properties of a novel oligopeptidase B from psychrotolerant gram-negative microorganism Serratia proteamaculans (PSP) were studied. The substrate specificity of PSP was analyzed using p-nitroanilide substrates, and the influence of calcium ions on the enzyme activity was studied. Hydrolysis of oligopeptides by PSP was studied using melittin as the substrate. Optimal conditions for the PSP activity (pH and temperature) have been established. It was found that PSP shares some properties with oligopeptidases B from other sources containing two Asp/Glu residues in the S2 site, but it differs significantly in some characteristics. The S2 site of PSP contains only one Asp460 residue. The secondary specificity of PSP has a number of specific features: an unusual substrate inhibition by peptides with hydrophobic residues at the P2 position, as well as the drastic influence of calcium ions on substrate characteristics of the enzyme. It is assumed that the PSP molecule contains a large hydrophobic substrate-binding site, and significant conformational rearrangements of the enzyme active site are induced by Ca²⁺ binding and by the formation of the enzyme–substrate complex. The temperature characteristics of PSP (high activity at low temperature as well as low apparent temperature optimum (25°C)) confirm that PSP is a psychrophilic enzyme.

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KEY WORDS: oligopeptidase B, Serratia proteamaculans, psychrophilic enzymes, substrate analysis, melittin, pH dependence

DOI: 10.1134/S0006297911040122

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