Structure-reactivity relationships for the inhibition mechanism at the second alkyl-chain-binding site of cholesterol esterase and lipase

Alkyl-N-phenyl carbamates (2-8) (see Figure 1), alkyl-N-phenyl thiocarbamat es (9-15), 2,2'-biphenyl-2-ol-2'-N-substituted carbamates (16-23), and 2, 2 '-biphenyl-2-N-octadecylcarbamate-2'-N-substituted carbamates (24-31) are p repared and evaluated for their inhibition effects on porcine pancreatic ch olesterol esterase and Pseudomona species lipase. All inhibitors are charac terized as transient or pseudo substrate inhibitors for both enzymes. Both enzymes are not protected from inhibition and further inactivated by carbam ates 2-8 and thiocarbamates 9-15 in the presence of trifluoroacetophenone. Therefore, carbamates 2-8 and thiocarbamates 9-15 are exceptions for active site binding inhibitors and are probably the second alkyl-chain binding-si te-directed inhibitors for both enzymes. The inhibition data for carbamates 2-8 and thiocarbamates 9-15 are correlated with the steric constant, E-s, and the hydrophobicity constant, pi; however, the inhibition data are not c orrelated with the Taft substituent constant, sigma*. A comparison of the i nhibition data for carbamates 2-8 and thiocarbamates 9-15 toward both enzym es indicates that thiocarbamates 9-15 are more potent inhibitors than carba mates 2-8. A comparison of the inhibition data for cholesterol esterase and Pseudomona species lipase by carbamates 2-8 or thiocarbamates 9-15 indicat es that cholesterol esterase is more sensitive to the E-s and pi values tha n Pseudomona species lipase. The negative slope values for the logarithms o f inhibition data for Pseudomona species lipase by carbamates 2-8 and thioc arbamates 9-15 versus E-s and pi indicate that the second alkyl-chain-bindi ng site of Pseudomona species lipase is huge, hydrophilic, compared to that of cholesterol esterase, and prefers to interact with a bulky, hydrophilic inhibitor rather than a small, hydrophobic one. On the contrary, the secon d alkyl-chain-binding site of cholesterol esterase prefers to bind to a sma ll, hydrophobic inhibitor. Both enzymes are protected from inhibition by ca rbamates 16-23 in the presence of trifluoroacetophenone. Therefore, carbama tes 16-23 are characterized as the alkyl chain binding site, esteratic site oxyanion active site directed pseudo substrate inhibitors for both enzymes . Both enzyme inhibition data for carbamates 16-22 are well-correlated with sigma* alone. The negative rho* values for these correlations indicate tha t the serine residue of both enzymes and carbamates 16-22 forms the tetrahe dral species with more positive charges than inhibitors and the enzymes and follow the formation of the carbamyl enzymes with more positive charges th an the tetrahedral species. Carbamates 24-31 are also exceptions for active site binding inhibitors and probably the second alkyl chain binding site-d irected inhibitors for both enzymes. However, the enzyme inhibition constan ts for carbamates 24-31 are correlated with values of sigma*, E-s, and pi. The negative rho* values for these correlations indicate that both enzymes and carbamates 24-31 form the tetrahedral species with more positive charge s than inhibitors and the enzymes and follow the formation of the carbamyl enzymes with more positive charges than those tetrahedral species. Therefor e, carbamates 24-31 may bind to both the active sites and the second alkyl chain binding site and follow the evacuation of the active sites. A compari son of the rho* values for cholesterol esterase and Pseudomona species lipa se by carbamates 24-31 indicates that cholesterol esterase is much more sen sitive to the sigma* values than Pseudomona species lipase. The negative sensitivity values, 9, for the cholesterol esterase inhibition s by carbamates 24-31 indicate that the enzyme prefers to bind to a bulky c arbamyl group rather than bind to a small one. The hydrophobicity of carbam ates 24-31 does not play a major role in both enzyme inhibitions.