Enhancement of the thermal stability of pyroglutamyl peptidase I by introduction of an intersubunit disulfide bond

doi:10.1016/S0167-4838(01)00185-6

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

Volume 1547, Issue 2, 11 June 2001, Pages 214-220

Biochimica et Biophy...

https://doi.org/10.1016/S0167-4838(01)00185-6 Get rights and content

Abstract

From the comparison of the three-dimensional structure of mesophilic pyroglutamyl peptidase from Bacillus amyloliquefaciens and the thermophilic enzyme from Thermococcus litoralis, the intersubunit disulfide bond was estimated to be one of the factors for thermal stability. Since Ser185 was corresponded to Cys190 of the thermophilic enzyme by sequence alignment, the Ser185 residue was replaced with cysteine by site-directed mutagenesis. The S185C mutant enzyme appeared to form a disulfide bond, which was confirmed by SDS–PAGE with and without 2-mercaptoethanol. The mutant enzyme showed a catalytic efficiency equivalent to that of the wild-type enzyme for hydrolysis of a synthetic peptide substrate. However, the thermal stability of the S185C mutant was found to be 30°C higher than that of wild-type. Thus the introduction of a disulfide bond enhanced thermal stability without changing the catalytic efficiency of the enzyme.

Introduction

Pyroglutamyl peptidase (PGP; EC 3.4.19.3) removes amino-terminal pyroglutamic acids (pGlu) from peptides and proteins. The enzymatic activity was previously reported to be present in several bacteria, plants, and animal tissues [1], [2], [3], [4], [5]. PGPs can be subdivided into two classes on a mechanistic basis. The type I class consists of mammalian and bacterial PGP that are all cysteine protease type enzymes. The type I enzyme degrades a broad spectrum of pGlu-containing peptides including thyrotropin releasing hormone (TRH), luteinizing hormone releasing hormone (LH-RH), bombesin, neurotensin and gastrins [6], [7]. The type II enzyme is an incompletely characterized mammalian protein that appears to be a membrane-bound metalloprotease localized in various tissues including muscle, cerebral cortex, and brain [6], [8], [9]. In contrast to the broad specificity of type I enzyme, type II enzyme is highly specific for TRH. Both classes of the mammalian enzymes have been implicated in the regulation of neuropeptide activity, for example in the TRH pathway [10], and in the neurotensin system [11]. Although the role of bacterial enzymes still remains unclear, it has been proposed that PGP activity serves to reduce the toxicity of N-terminally blocked peptides [6], or plays a role in nutrient assimilation [1].

We reported the cloning of the PGP gene from Bacillus amyloliquefaciens (BPGP), characterization of its enzymatic properties [12], the three-dimensional structure of the enzyme (Fig. 1 (1)) [13], and the mechanism of substrate recognition [14].

Recently, Singleton et al. reported the crystal structure of the PGP from the hyperthermophilic archaeon Thermococcus litoralis (TPGP) [15]. TPGP showed enhanced thermal stability when compared with enzymes from mesophilic bacteria. TPGP is also a homotetramer, and its three-dimensional structure is similar to that of BPGP (Fig. 1 (2)). However, there were two disulfide bonds between the subunits (A-ss-B, C-ss-D) in TPGP, but not in BPGP. This intersubunit disulfide bond seems to be responsible for the thermal stability of the TPGP. We found that the introduction of the intersubunit disulfide bond enhanced the thermal stability of BPGP without any change in its catalytic efficiency.

Section snippets

Materials

Restriction endonucleases and various DNA-modifying enzymes were obtained from New England Biolabs or Toyobo Biochemicals. Pyroglutamyl-β-naphthylamide (pGlu-2NNap) and Fast Garnet GBC were from Sigma. The oligonucleotide primer for site-directed mutagenesis was synthesized by Amersham Pharmacia Biotech. The Mutan-Super Express Km kit and plasmid pKF18k were from Takara Shuzo. The ALF express AutoCycle sequencing kit and other reagents for DNA sequencing were obtained from Amersham Pharmacia

Intersubunit disulfide bond formation

From the comparison of the three-dimensional structure between BPGP [13] and TPGP [15], it was assumed that the higher thermal stability of TPGP arises from the presence of intersubunit disulfide bridges. These two enzymes are homotetramers, and their three-dimensional structures are similar. However, an intersubunit disulfide bond is formed from the Cys190 in TPGP, while it did not exist in BPGP. Amino acid sequence alignment around the Cys190 of TPGP is shown in Fig. 2. Since the Ser185

References (24)

W.L. Taylor et al.
J. Biol. Chem.
(1978)
K. Fujiwara et al.
Biochim. Biophys. Acta
(1979)
J. Boler et al.
Biochem. Biophys. Res. Commun.
(1969)
Y. Odagaki et al.
Structure
(1999)
M. Singleton et al.
Structure
(1999)
T. Hashimoto-Gotoh et al.
Gene
(1995)
C.N. Pace et al.
J. Biol. Chem.
(1988)
H. Takagi et al.
J. Biol. Chem.
(1990)
J.H. Ko et al.
Biochem. Biophys. Res. Commun.
(1996)
J. Mansfeld et al.
J. Biol. Chem.
(1997)

R.F. Doolittle et al.

Biochemistry

(1968)

A. Szewczuk et al.

Eur. J. Biochem.

(1969)

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Enhancement of the thermal stability of pyroglutamyl peptidase I by introduction of an intersubunit disulfide bond

Abstract

Introduction

Section snippets

Materials

Intersubunit disulfide bond formation

J. Biol. Chem.

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

Structure

Structure

Gene

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochemistry

Eur. J. Biochem.