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Biochemistry (Moscow)

, 76:480 | Cite as

Oligopeptidase B from Serratia proteamaculans. II. Enzymatic characteristics: Substrate analysis, influence of calcium ions, pH and temperature dependences

  • A. G. MikhailovaEmail author
  • R. F. Khairullin
  • I. V. Demidyuk
  • T. Yu. Gromova
  • S. V. Kostrov
  • L. D. Rumsh
Article

Abstract

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.

Key words

oligopeptidase B Serratia proteamaculans psychrophilic enzymes substrate analysis melittin pH dependence 

Abbreviations

BAPNA

Nα-benzoyl-DL-arginine-p-nitroanilide

buffer A

0.1 M Tris-HCl, pH 8.0

buffer B

0.1 M Tris-HCl, pH 8.0, 50 mM CaCl2

Bz

benzoyl

DMSO

dimethyl sulfoxide

OpdB

oligopeptidase B

p-NA

p-nitroanilide

PSP

oligopeptidase B from Serratia proteamaculans

Suc

succinyl

Z

benzyloxycarbonyl

Z-Lys-S-Bzl

Nα-benzyloxycarbonyl-L-lysine thiobenzyl ester

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Copyright information

© Pleiades Publishing, Ltd. 2011

Authors and Affiliations

  • A. G. Mikhailova
    • 1
    Email author
  • R. F. Khairullin
    • 1
  • I. V. Demidyuk
    • 2
  • T. Yu. Gromova
    • 2
  • S. V. Kostrov
    • 2
  • L. D. Rumsh
    • 1
  1. 1.Shemyakin and Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussia
  2. 2.Institute of Molecular GeneticsRussian Academy of SciencesMoscowRussia

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