Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli

Weilie Zhang, Qicun Shi, Samy Meroueh, Sergei B. Vakulenko, Shahriar Mobashery

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

Penicillin-binding proteins (PBPs) and β-lactamases are members of large families of bacterial enzymes. These enzymes undergo acylation at a serine residue with their respective substrates as the first step in their catalytic events. Penicillin-binding protein 5 (PBP 5) of Escherichia coli is known to perform a DD-carboxypeptidase reaction on the bacterial peptidoglycan, the major constituent of the cell wall. The roles of the active site residues Lys47 and Lys213 in the catalytic machinery of PBP 5 have been explored. By a sequence of site-directed mutagenesis and chemical modification, we individually introduced γ-thialysine at each of these positions. The pH dependence of k cat/Km and of kcat for the wild-type PBP 5 and for the two γ-thialysine mutant variants at positions 47 and 213 were evaluated. The pH optimum for the enzyme was at 9.5-10.5. The ascending limb to the pH optimum is due to Lys47; hence, this residue exists in the free-base form for catalysis. The descending limb from the pH optimum is contributed to by both Lys213 and a water molecule coordinated to Lys47. These results have been interpreted as Lys47 playing a key role in proton-transfer events in the course of catalysis during both the acylation and deacylation events. However, the findings for Lys213 argue for a protonated state at the pH optimum. Lys213 serves as an electrostatic anchor for the substrate.

Original languageEnglish (US)
Pages (from-to)10113-10121
Number of pages9
JournalBiochemistry
Volume46
Issue number35
DOIs
StatePublished - Sep 4 2007
Externally publishedYes

Fingerprint

Penicillin-Binding Proteins
Escherichia coli
Acylation
Catalysis
Enzymes
Carboxypeptidases
Extremities
Mutagenesis
Proton transfer
Peptidoglycan
Chemical modification
Substrates
Anchors
Serine
Machinery
Electrostatics
Site-Directed Mutagenesis
Static Electricity
Cell Wall
Cells

ASJC Scopus subject areas

  • Biochemistry

Cite this

Zhang, W., Shi, Q., Meroueh, S., Vakulenko, S. B., & Mobashery, S. (2007). Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli. Biochemistry, 46(35), 10113-10121. https://doi.org/10.1021/bi700777x

Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli. / Zhang, Weilie; Shi, Qicun; Meroueh, Samy; Vakulenko, Sergei B.; Mobashery, Shahriar.

In: Biochemistry, Vol. 46, No. 35, 04.09.2007, p. 10113-10121.

Research output: Contribution to journalArticle

Zhang, W, Shi, Q, Meroueh, S, Vakulenko, SB & Mobashery, S 2007, 'Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli', Biochemistry, vol. 46, no. 35, pp. 10113-10121. https://doi.org/10.1021/bi700777x
Zhang, Weilie ; Shi, Qicun ; Meroueh, Samy ; Vakulenko, Sergei B. ; Mobashery, Shahriar. / Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli. In: Biochemistry. 2007 ; Vol. 46, No. 35. pp. 10113-10121.
@article{7a7f9f0d6e3549b0b7d854c7f4158260,
title = "Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli",
abstract = "Penicillin-binding proteins (PBPs) and β-lactamases are members of large families of bacterial enzymes. These enzymes undergo acylation at a serine residue with their respective substrates as the first step in their catalytic events. Penicillin-binding protein 5 (PBP 5) of Escherichia coli is known to perform a DD-carboxypeptidase reaction on the bacterial peptidoglycan, the major constituent of the cell wall. The roles of the active site residues Lys47 and Lys213 in the catalytic machinery of PBP 5 have been explored. By a sequence of site-directed mutagenesis and chemical modification, we individually introduced γ-thialysine at each of these positions. The pH dependence of k cat/Km and of kcat for the wild-type PBP 5 and for the two γ-thialysine mutant variants at positions 47 and 213 were evaluated. The pH optimum for the enzyme was at 9.5-10.5. The ascending limb to the pH optimum is due to Lys47; hence, this residue exists in the free-base form for catalysis. The descending limb from the pH optimum is contributed to by both Lys213 and a water molecule coordinated to Lys47. These results have been interpreted as Lys47 playing a key role in proton-transfer events in the course of catalysis during both the acylation and deacylation events. However, the findings for Lys213 argue for a protonated state at the pH optimum. Lys213 serves as an electrostatic anchor for the substrate.",
author = "Weilie Zhang and Qicun Shi and Samy Meroueh and Vakulenko, {Sergei B.} and Shahriar Mobashery",
year = "2007",
month = "9",
day = "4",
doi = "10.1021/bi700777x",
language = "English (US)",
volume = "46",
pages = "10113--10121",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "35",

}

TY - JOUR

T1 - Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli

AU - Zhang, Weilie

AU - Shi, Qicun

AU - Meroueh, Samy

AU - Vakulenko, Sergei B.

AU - Mobashery, Shahriar

PY - 2007/9/4

Y1 - 2007/9/4

N2 - Penicillin-binding proteins (PBPs) and β-lactamases are members of large families of bacterial enzymes. These enzymes undergo acylation at a serine residue with their respective substrates as the first step in their catalytic events. Penicillin-binding protein 5 (PBP 5) of Escherichia coli is known to perform a DD-carboxypeptidase reaction on the bacterial peptidoglycan, the major constituent of the cell wall. The roles of the active site residues Lys47 and Lys213 in the catalytic machinery of PBP 5 have been explored. By a sequence of site-directed mutagenesis and chemical modification, we individually introduced γ-thialysine at each of these positions. The pH dependence of k cat/Km and of kcat for the wild-type PBP 5 and for the two γ-thialysine mutant variants at positions 47 and 213 were evaluated. The pH optimum for the enzyme was at 9.5-10.5. The ascending limb to the pH optimum is due to Lys47; hence, this residue exists in the free-base form for catalysis. The descending limb from the pH optimum is contributed to by both Lys213 and a water molecule coordinated to Lys47. These results have been interpreted as Lys47 playing a key role in proton-transfer events in the course of catalysis during both the acylation and deacylation events. However, the findings for Lys213 argue for a protonated state at the pH optimum. Lys213 serves as an electrostatic anchor for the substrate.

AB - Penicillin-binding proteins (PBPs) and β-lactamases are members of large families of bacterial enzymes. These enzymes undergo acylation at a serine residue with their respective substrates as the first step in their catalytic events. Penicillin-binding protein 5 (PBP 5) of Escherichia coli is known to perform a DD-carboxypeptidase reaction on the bacterial peptidoglycan, the major constituent of the cell wall. The roles of the active site residues Lys47 and Lys213 in the catalytic machinery of PBP 5 have been explored. By a sequence of site-directed mutagenesis and chemical modification, we individually introduced γ-thialysine at each of these positions. The pH dependence of k cat/Km and of kcat for the wild-type PBP 5 and for the two γ-thialysine mutant variants at positions 47 and 213 were evaluated. The pH optimum for the enzyme was at 9.5-10.5. The ascending limb to the pH optimum is due to Lys47; hence, this residue exists in the free-base form for catalysis. The descending limb from the pH optimum is contributed to by both Lys213 and a water molecule coordinated to Lys47. These results have been interpreted as Lys47 playing a key role in proton-transfer events in the course of catalysis during both the acylation and deacylation events. However, the findings for Lys213 argue for a protonated state at the pH optimum. Lys213 serves as an electrostatic anchor for the substrate.

UR - http://www.scopus.com/inward/record.url?scp=34548473816&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34548473816&partnerID=8YFLogxK

U2 - 10.1021/bi700777x

DO - 10.1021/bi700777x

M3 - Article

C2 - 17685588

AN - SCOPUS:34548473816

VL - 46

SP - 10113

EP - 10121

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 35

ER -