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|Title:||Factors affecting the reaction of lecithin cholesterol acyltransferase with water-soluble substrates|
|Author(s):||Bonelli, Frank Scott|
|Doctoral Committee Chair(s):||Jonas, Ana|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||To separate interfacial binding from the catalytic reactions of LCAT, the first investigation of its reaction with water soluble substrates was performed to characterize the molecular specificity of the active site as well as the effects of external modulators on the enzyme. Two substrates were chosen: fatty acid esters of p-nitrophenol and 1,2-Bis (4-(1-pyreno)butanoyl) -sn-glycero-3-phosphocholine (DPydPC).
The fatty acid esters of p-nitrophenol were used to determine the chain length specificity of LCAT. The kinetics of the LCAT reaction were measured for the PNP esters with fatty acids having up to six (C-6) carbons in length. With increasing acyl chain lengths, the initial velocity of PNP ester hydrolysis went through a maximum at the C-5 esters. The Km and Vmax values decreased progressively, while the kcat/km values increased with increasing acyl chain length.
Both water soluble substrates were used to study the effects of modulators of the LCAT reaction (e.g., lysolecithin, salt concentration, and apolipoprotein A-I) on the enzyme itself. Inhibition of the LCAT reaction by lysolecithin, fatty acids, and detergents of the hydrolysis of the water soluble substrates by LCAT indicated that the effects of these compounds are due to a direct interaction with the enzyme.
The effects of anions, cations, and increasing salt concentration had no effect on the hydrolysis of the water soluble substrates, in contrast to their effects on the LCAT reaction in the presence of an interface. Thus, these effects are mediated primarily on the interaction of the enzyme with the interface. The involvement of cationic residues on LCAT in interfacial binding is also postulated.
Hydrolysis of the water soluble substrates occurred at high reaction rates relative to rHDL containing apolipoprotein A-I. This observation as well as the fact that solubilized apolipoproteins do not affect hydrolysis of the water soluble substrates suggests that activation of the LCAT reaction by apolipoprotein A-I also is exclusively an interfacial effect.
|Rights Information:||Copyright 1990 Bonelli, Frank Scott|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9021653|
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