Oligopeptidase B: A new type of serine peptidase with a unique substrate-dependent temperature sensitivity

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Abstract

Oligopeptidase B, a member of the novel prolyl oligopeptidase family of serine peptidases, in involved in cell invasion by trypanosomes. The kinetic analysis of the reactions of oligopeptidase B, which preferentially cleaves peptides at two adjacent basic residues, has revealed significant differences from the trypsin-like serine peptidases. (i) The pH dependence of k(cat)/K(m) deviates from normal bell-shaped curves due to ionization of an enzymatic group characterized by a macroscopic pK(a) of ~8.3. The effect of this group is abolished at high ionic strength. (ii) The second-order acylation rate constants, k(cat)/K(m), are similar with the ester and the corresponding amide substrates, suggesting that their chemical reactivity does not prevail in the rate-limiting step. The kinetic deuterium isotope effects indicate that the rate-limiting step for k(cat)/K(m) is principally governed by conformational changes. (iii) The pH-k(cat)/K(m) profile and the very low rate constant for benzoyl-citrulline ethyl ester reveal a new kinetically influential group ionizing below the pK(a) of the active site histidine and indicate that the positive charge of arginine is essential for effective catalysis. (iv) The enzyme is inhibited by high concentrations of substrate. The mechanism of inhibition markedly varies with the reaction conditions. (v) The optimum temperature for the reactions of amide substrates is unusually low, slightly below 25 °C, whereas with benzoyl-arginine ethyl ester a linear Eyring plot is obtained up to 39 °C. The positive entropies of activation point to substantial reorganization of water molecules upon substrate binding.

Original languageEnglish
Pages (from-to)15548-15555
Number of pages8
JournalBiochemistry
Volume38
Issue number47
DOIs
Publication statusPublished - Nov 23 1999

Fingerprint

oligopeptidase B
Amides
Serine
prolyl oligopeptidase
Esters
Peptide Hydrolases
Citrulline
Temperature
Acylation
Trypanosomiasis
Deuterium
Entropy
Substrates
Catalysis
Histidine
Isotopes
Osmolar Concentration
Trypsin
Arginine
Rate constants

ASJC Scopus subject areas

  • Biochemistry

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Oligopeptidase B : A new type of serine peptidase with a unique substrate-dependent temperature sensitivity. / Polgár, L.

In: Biochemistry, Vol. 38, No. 47, 23.11.1999, p. 15548-15555.

Research output: Contribution to journalArticle

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abstract = "Oligopeptidase B, a member of the novel prolyl oligopeptidase family of serine peptidases, in involved in cell invasion by trypanosomes. The kinetic analysis of the reactions of oligopeptidase B, which preferentially cleaves peptides at two adjacent basic residues, has revealed significant differences from the trypsin-like serine peptidases. (i) The pH dependence of k(cat)/K(m) deviates from normal bell-shaped curves due to ionization of an enzymatic group characterized by a macroscopic pK(a) of ~8.3. The effect of this group is abolished at high ionic strength. (ii) The second-order acylation rate constants, k(cat)/K(m), are similar with the ester and the corresponding amide substrates, suggesting that their chemical reactivity does not prevail in the rate-limiting step. The kinetic deuterium isotope effects indicate that the rate-limiting step for k(cat)/K(m) is principally governed by conformational changes. (iii) The pH-k(cat)/K(m) profile and the very low rate constant for benzoyl-citrulline ethyl ester reveal a new kinetically influential group ionizing below the pK(a) of the active site histidine and indicate that the positive charge of arginine is essential for effective catalysis. (iv) The enzyme is inhibited by high concentrations of substrate. The mechanism of inhibition markedly varies with the reaction conditions. (v) The optimum temperature for the reactions of amide substrates is unusually low, slightly below 25 °C, whereas with benzoyl-arginine ethyl ester a linear Eyring plot is obtained up to 39 °C. The positive entropies of activation point to substantial reorganization of water molecules upon substrate binding.",
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