Preparative Enzymatic Synthesis of Acyl-Acyl Carrier Protein

Charles O. Rock, Jeffrey L. Garwin, John E Cronan

Research output: Contribution to journalArticle

Abstract

This chapter discusses the preparative enzymatic synthesis of acyl-acyl carrier protein. Fatty acids are synthesized de novo as thioesters of acyl carrier protein (ACP), and long-chain acyl-ACPs serve as substrates for the snglycerol-3-phosphate acyltransferase in Escherichia coli. The most widely used chemical procedure results in preparations of acyl-ACP possessing highly variable biological activity, due to acetylation of the ε-amino groups of the four lysine residues and the NH2-terminal serine. Acetylation has been shown to result in a dramatic loss of secondary structure and alteration of the biological activity of ACP-SH and of acyl-ACP. It is found that when large amounts of acyl-ACP are being prepared it is useful to recover acyl-ACP synthetase from the incubation mixtures for use in further syntheses. If acyl-ACP synthetase is not going to be recovered, this step can be omitted and the incubation mixture applied directly to DEAE-cellulose. Acyl-ACP is much more stable to base-catalyzed hydrolysis than the analogous acyl-CoA 7. This property allows experiments with acyl-ACP at pH values up to 10.0 to be performed without significant hydrolysis occurring. The presence of the acyl moiety on ACP also stabilizes the protein to pH-induced hydrodynamic expansion, permitting the separation of ACP from acyl-ACP on gel filtration columns.

Original languageEnglish (US)
Pages (from-to)397-403
Number of pages7
JournalMethods in enzymology
Volume72
Issue numberC
DOIs
StatePublished - Jan 1 1981
Externally publishedYes

Fingerprint

Acyl Carrier Protein
Acetylation
Bioactivity
Hydrolysis
Acyltransferases
DEAE-Cellulose
Acyl Coenzyme A
Hydrodynamics
Serine
Escherichia coli
Lysine
Gel Chromatography
Fatty Acids

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Preparative Enzymatic Synthesis of Acyl-Acyl Carrier Protein. / Rock, Charles O.; Garwin, Jeffrey L.; Cronan, John E.

In: Methods in enzymology, Vol. 72, No. C, 01.01.1981, p. 397-403.

Research output: Contribution to journalArticle

Rock, Charles O. ; Garwin, Jeffrey L. ; Cronan, John E. / Preparative Enzymatic Synthesis of Acyl-Acyl Carrier Protein. In: Methods in enzymology. 1981 ; Vol. 72, No. C. pp. 397-403.
@article{f7cc63d1c3024f8c85caff251fd9b355,
title = "Preparative Enzymatic Synthesis of Acyl-Acyl Carrier Protein",
abstract = "This chapter discusses the preparative enzymatic synthesis of acyl-acyl carrier protein. Fatty acids are synthesized de novo as thioesters of acyl carrier protein (ACP), and long-chain acyl-ACPs serve as substrates for the snglycerol-3-phosphate acyltransferase in Escherichia coli. The most widely used chemical procedure results in preparations of acyl-ACP possessing highly variable biological activity, due to acetylation of the ε-amino groups of the four lysine residues and the NH2-terminal serine. Acetylation has been shown to result in a dramatic loss of secondary structure and alteration of the biological activity of ACP-SH and of acyl-ACP. It is found that when large amounts of acyl-ACP are being prepared it is useful to recover acyl-ACP synthetase from the incubation mixtures for use in further syntheses. If acyl-ACP synthetase is not going to be recovered, this step can be omitted and the incubation mixture applied directly to DEAE-cellulose. Acyl-ACP is much more stable to base-catalyzed hydrolysis than the analogous acyl-CoA 7. This property allows experiments with acyl-ACP at pH values up to 10.0 to be performed without significant hydrolysis occurring. The presence of the acyl moiety on ACP also stabilizes the protein to pH-induced hydrodynamic expansion, permitting the separation of ACP from acyl-ACP on gel filtration columns.",
author = "Rock, {Charles O.} and Garwin, {Jeffrey L.} and Cronan, {John E}",
year = "1981",
month = "1",
day = "1",
doi = "10.1016/S0076-6879(81)72029-9",
language = "English (US)",
volume = "72",
pages = "397--403",
journal = "Methods in Enzymology",
issn = "0076-6879",
publisher = "Academic Press Inc.",
number = "C",

}

TY - JOUR

T1 - Preparative Enzymatic Synthesis of Acyl-Acyl Carrier Protein

AU - Rock, Charles O.

AU - Garwin, Jeffrey L.

AU - Cronan, John E

PY - 1981/1/1

Y1 - 1981/1/1

N2 - This chapter discusses the preparative enzymatic synthesis of acyl-acyl carrier protein. Fatty acids are synthesized de novo as thioesters of acyl carrier protein (ACP), and long-chain acyl-ACPs serve as substrates for the snglycerol-3-phosphate acyltransferase in Escherichia coli. The most widely used chemical procedure results in preparations of acyl-ACP possessing highly variable biological activity, due to acetylation of the ε-amino groups of the four lysine residues and the NH2-terminal serine. Acetylation has been shown to result in a dramatic loss of secondary structure and alteration of the biological activity of ACP-SH and of acyl-ACP. It is found that when large amounts of acyl-ACP are being prepared it is useful to recover acyl-ACP synthetase from the incubation mixtures for use in further syntheses. If acyl-ACP synthetase is not going to be recovered, this step can be omitted and the incubation mixture applied directly to DEAE-cellulose. Acyl-ACP is much more stable to base-catalyzed hydrolysis than the analogous acyl-CoA 7. This property allows experiments with acyl-ACP at pH values up to 10.0 to be performed without significant hydrolysis occurring. The presence of the acyl moiety on ACP also stabilizes the protein to pH-induced hydrodynamic expansion, permitting the separation of ACP from acyl-ACP on gel filtration columns.

AB - This chapter discusses the preparative enzymatic synthesis of acyl-acyl carrier protein. Fatty acids are synthesized de novo as thioesters of acyl carrier protein (ACP), and long-chain acyl-ACPs serve as substrates for the snglycerol-3-phosphate acyltransferase in Escherichia coli. The most widely used chemical procedure results in preparations of acyl-ACP possessing highly variable biological activity, due to acetylation of the ε-amino groups of the four lysine residues and the NH2-terminal serine. Acetylation has been shown to result in a dramatic loss of secondary structure and alteration of the biological activity of ACP-SH and of acyl-ACP. It is found that when large amounts of acyl-ACP are being prepared it is useful to recover acyl-ACP synthetase from the incubation mixtures for use in further syntheses. If acyl-ACP synthetase is not going to be recovered, this step can be omitted and the incubation mixture applied directly to DEAE-cellulose. Acyl-ACP is much more stable to base-catalyzed hydrolysis than the analogous acyl-CoA 7. This property allows experiments with acyl-ACP at pH values up to 10.0 to be performed without significant hydrolysis occurring. The presence of the acyl moiety on ACP also stabilizes the protein to pH-induced hydrodynamic expansion, permitting the separation of ACP from acyl-ACP on gel filtration columns.

UR - http://www.scopus.com/inward/record.url?scp=0019765709&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0019765709&partnerID=8YFLogxK

U2 - 10.1016/S0076-6879(81)72029-9

DO - 10.1016/S0076-6879(81)72029-9

M3 - Article

C2 - 7031424

AN - SCOPUS:0019765709

VL - 72

SP - 397

EP - 403

JO - Methods in Enzymology

JF - Methods in Enzymology

SN - 0076-6879

IS - C

ER -