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 language | English (US) |
|---|---|
| Pages (from-to) | 397-403 |
| Number of pages | 7 |
| Journal | Methods in enzymology |
| Volume | 72 |
| Issue number | C |
| DOIs | |
| State | Published - Jan 1 1981 |
| Externally published | Yes |
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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 journal › Article
}
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.
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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 -