sn-1,2-diacylglycerol kinase of Escherichia coli. Structural and kinetic analysis of the lipid cofactor dependence

J. P. Walsh, R. M. Bell

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

The lipid cofactor requirement of Escherichia coli sn-1,2-diacylglycerol kinase was studied using a β-octylglucoside mixed micellar assay (Walsh, J.P., and Bell, R.M. (1986) J. Biol. Chem. 261, 6239-6247). The enzyme was shown to have an absolute requirement for a lipid activator. sn-1,2-Dioleoylglycerol was both an activator and a substrate for the enzyme, 1,3-dioleoylglycerol was an activator but not a substrate, and sn-1,2-dioctanoylglycerol was a substrate but not an activator. Activation was observed with a large number of phospholipids, sulfolipids, neutral lipids, and detergents. Lipids with longer alkyl/acyl chains stimulated activity to a greater extent and lower concentrations than their shorter chain homologs. Anionic lipids were the best activators, and neutral lipids were somewhat less effective. Cationic lipids were poor activators. Lipid activation was cooperative in all cases, with Hill coefficients ranging from 2.9 to 4.7. Lipid activators stabilized the enzyme against inactivation induced by diacylglycerols. The effectiveness of several lipids in stabilizing the enzyme correlated with their effectiveness as kinetic activators, suggesting a common mechanism. Kinetic analyses also suggested that a lipid cofactor-induced conformational change occurs as a part of the activation process. β-Octylglucoside was shown not to function as a lipid cofactor for diacylglycerol kinase. The requirement for detergent in the assay was related, instead, to the need to disperse and deliver water-insoluble substrates and cofactors to the enzyme. β-Octyglucoside also provided an inert matrix to which lipid substrates and cofactors could be added, enabling study of their concentration dependencies.

Original languageEnglish (US)
Pages (from-to)15062-15069
Number of pages8
JournalJournal of Biological Chemistry
Volume261
Issue number32
StatePublished - 1986
Externally publishedYes

Fingerprint

Diacylglycerol Kinase
Escherichia coli
Lipids
Kinetics
Substrates
Enzymes
Chemical activation
Detergents
Assays
Enzyme Activators
Diglycerides
Coenzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

sn-1,2-diacylglycerol kinase of Escherichia coli. Structural and kinetic analysis of the lipid cofactor dependence. / Walsh, J. P.; Bell, R. M.

In: Journal of Biological Chemistry, Vol. 261, No. 32, 1986, p. 15062-15069.

Research output: Contribution to journalArticle

@article{03c139efca894e9e8de1835631293461,
title = "sn-1,2-diacylglycerol kinase of Escherichia coli. Structural and kinetic analysis of the lipid cofactor dependence",
abstract = "The lipid cofactor requirement of Escherichia coli sn-1,2-diacylglycerol kinase was studied using a β-octylglucoside mixed micellar assay (Walsh, J.P., and Bell, R.M. (1986) J. Biol. Chem. 261, 6239-6247). The enzyme was shown to have an absolute requirement for a lipid activator. sn-1,2-Dioleoylglycerol was both an activator and a substrate for the enzyme, 1,3-dioleoylglycerol was an activator but not a substrate, and sn-1,2-dioctanoylglycerol was a substrate but not an activator. Activation was observed with a large number of phospholipids, sulfolipids, neutral lipids, and detergents. Lipids with longer alkyl/acyl chains stimulated activity to a greater extent and lower concentrations than their shorter chain homologs. Anionic lipids were the best activators, and neutral lipids were somewhat less effective. Cationic lipids were poor activators. Lipid activation was cooperative in all cases, with Hill coefficients ranging from 2.9 to 4.7. Lipid activators stabilized the enzyme against inactivation induced by diacylglycerols. The effectiveness of several lipids in stabilizing the enzyme correlated with their effectiveness as kinetic activators, suggesting a common mechanism. Kinetic analyses also suggested that a lipid cofactor-induced conformational change occurs as a part of the activation process. β-Octylglucoside was shown not to function as a lipid cofactor for diacylglycerol kinase. The requirement for detergent in the assay was related, instead, to the need to disperse and deliver water-insoluble substrates and cofactors to the enzyme. β-Octyglucoside also provided an inert matrix to which lipid substrates and cofactors could be added, enabling study of their concentration dependencies.",
author = "Walsh, {J. P.} and Bell, {R. M.}",
year = "1986",
language = "English (US)",
volume = "261",
pages = "15062--15069",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "32",

}

TY - JOUR

T1 - sn-1,2-diacylglycerol kinase of Escherichia coli. Structural and kinetic analysis of the lipid cofactor dependence

AU - Walsh, J. P.

AU - Bell, R. M.

PY - 1986

Y1 - 1986

N2 - The lipid cofactor requirement of Escherichia coli sn-1,2-diacylglycerol kinase was studied using a β-octylglucoside mixed micellar assay (Walsh, J.P., and Bell, R.M. (1986) J. Biol. Chem. 261, 6239-6247). The enzyme was shown to have an absolute requirement for a lipid activator. sn-1,2-Dioleoylglycerol was both an activator and a substrate for the enzyme, 1,3-dioleoylglycerol was an activator but not a substrate, and sn-1,2-dioctanoylglycerol was a substrate but not an activator. Activation was observed with a large number of phospholipids, sulfolipids, neutral lipids, and detergents. Lipids with longer alkyl/acyl chains stimulated activity to a greater extent and lower concentrations than their shorter chain homologs. Anionic lipids were the best activators, and neutral lipids were somewhat less effective. Cationic lipids were poor activators. Lipid activation was cooperative in all cases, with Hill coefficients ranging from 2.9 to 4.7. Lipid activators stabilized the enzyme against inactivation induced by diacylglycerols. The effectiveness of several lipids in stabilizing the enzyme correlated with their effectiveness as kinetic activators, suggesting a common mechanism. Kinetic analyses also suggested that a lipid cofactor-induced conformational change occurs as a part of the activation process. β-Octylglucoside was shown not to function as a lipid cofactor for diacylglycerol kinase. The requirement for detergent in the assay was related, instead, to the need to disperse and deliver water-insoluble substrates and cofactors to the enzyme. β-Octyglucoside also provided an inert matrix to which lipid substrates and cofactors could be added, enabling study of their concentration dependencies.

AB - The lipid cofactor requirement of Escherichia coli sn-1,2-diacylglycerol kinase was studied using a β-octylglucoside mixed micellar assay (Walsh, J.P., and Bell, R.M. (1986) J. Biol. Chem. 261, 6239-6247). The enzyme was shown to have an absolute requirement for a lipid activator. sn-1,2-Dioleoylglycerol was both an activator and a substrate for the enzyme, 1,3-dioleoylglycerol was an activator but not a substrate, and sn-1,2-dioctanoylglycerol was a substrate but not an activator. Activation was observed with a large number of phospholipids, sulfolipids, neutral lipids, and detergents. Lipids with longer alkyl/acyl chains stimulated activity to a greater extent and lower concentrations than their shorter chain homologs. Anionic lipids were the best activators, and neutral lipids were somewhat less effective. Cationic lipids were poor activators. Lipid activation was cooperative in all cases, with Hill coefficients ranging from 2.9 to 4.7. Lipid activators stabilized the enzyme against inactivation induced by diacylglycerols. The effectiveness of several lipids in stabilizing the enzyme correlated with their effectiveness as kinetic activators, suggesting a common mechanism. Kinetic analyses also suggested that a lipid cofactor-induced conformational change occurs as a part of the activation process. β-Octylglucoside was shown not to function as a lipid cofactor for diacylglycerol kinase. The requirement for detergent in the assay was related, instead, to the need to disperse and deliver water-insoluble substrates and cofactors to the enzyme. β-Octyglucoside also provided an inert matrix to which lipid substrates and cofactors could be added, enabling study of their concentration dependencies.

UR - http://www.scopus.com/inward/record.url?scp=0023035961&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0023035961&partnerID=8YFLogxK

M3 - Article

VL - 261

SP - 15062

EP - 15069

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 32

ER -