Binding of pancreatic carboxylester lipase to mixed lipid films. Implications for surface organization.
The hydrolysis of ester substrates in mixed monolayers with 1-palmitoyl-2-oleoylphosphatidylcholine by pancreatic carboxylester lipase (CEL) shows an abrupt increase from less than 10 to 100% when substrate abundance exceeds a critical value (Tsujita, T., Muderhwa, J.M., and Brockman, H.L. (1989) J. Biol. Chem. 264, 8612-8618). Adsorption of native CEL to these mixed lipid films under the same conditions shows a monotonic increase with substrate abundance to a maximum of approximately 4 pmol/cm2. In the range of low substrate hydrolysis, bound enzyme reaches 0.6 pmol/cm2 or about one-sixth of an enzyme monolayer. Given the high turnover number of the enzyme, absence of bound catalyst cannot explain the lack of substrate hydrolysis. Similarities of the adsorption data obtained with different substrates suggest a common, area-based mechanism with an excluded area for the phospholipid. Quantitative analysis confirms this and gives an excluded area of 43.5 +/- 1.0 A2/molecule. Comparable studies with the hydrolysis product, oleic acid, replacing each substrate show markedly different results. Up to oleic acid mol fractions of 0.5, bound CEL levels are essentially constant at 0.215 pmol/cm2. This behavior relative to substrate-containing films cannot be ascribed to differences in surface pressure, surface potential, monolayer compressibility, nonexcluded area, CEL denaturation, or oleic acid charge. Together with the ultrasensitive composition dependence of substrate hydrolysis, the data suggest that phospholipid-based organization of the surface is a primary regulator of enzyme binding and catalysis.
Muderhwa
J M
JM
Hormel Institute, University of Minnesota, Austin 55912.
Brockman
H L
HL
D000327:Adsorption; D000818:Animals; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D006868:Hydrolysis; D050356:Lipid Metabolism; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000378:metabolism; D013499:Surface Properties; D013552:Swine; D014280:Triglycerides / Q000378:metabolism
D004075:Diglycerides; D005227:Fatty Acids; D009829:Oleic Acids; D010713:Phosphatidylcholines; D014280:Triglycerides; D019301:Oleic Acid; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; C028694:1-palmitoyl-2-oleoylphosphatidylcholine