Title : Molecular recognition by cholesterol esterase of active site ligands: structure-reactivity effects for inhibition by aryl carbamates and subsequent carbamylenzyme turnover - Feaster_1996_Biochemistry_35_16723 |
Author(s) : Feaster SR , Lee K , Baker N , Hui DY , Quinn DM |
Ref : Biochemistry , 35 :16723 , 1996 |
Abstract :
Interactions of mammalian pancreatic cholesterol esterases from pig and rat with a family of aryl carbamates CnH2n+1NHCOOAr [n = 4-9; Ar = phenyl, p-X-phenyl (X = acetamido, bromo, fluoro, nitro, trifluoromethyl), 2-naphthyl, 2-tetrahydronaphthyl, estronyl] have been investigated, with an aim of delineating the ligand structural features which lead to effective molecular recognition by the active site of the enzyme. These carbamates inhibit the catalytic activity of CEase by rapid carbamylation of the active site, a process that shows saturation kinetics. Subsequent slow decarbamylation usually leads to full restoration of activity, and therefore aryl carbamates are transient inhibitors, or pseudo-substrates, of CEase. Structural variation of carbamate inhibitors allowed molecular recognition in the fatty acid binding and steroid binding loci of the extended active site to be probed, and the electronic nature of the carbamylation transition state to be characterized. Optimal inhibitory activity is observed when the length of the carbamyl function is n = 6 and n = 7 for porcine and rat cholesterol esterases, respectively, equivalent to eight- and nine-carbon fatty acyl chains. In contrast, inhibitory activity increases progressively as the partial molecular volume of the aromatic fragment increases. Hammett plots for p-substituted phenyl-N-hexyl carbamates indicate that the rate-determining step for carbamate inhibition is phenolate anion expulsion. Effects of the bile salt activator taurocholate on the kinetically resolved phases of the pseudo-substrate turnover of aryl carbamates were also studied. Taurocholate increases the affinity of the carbamate for the active site of cholesterol esterase in the reversible, noncovalent complex that precedes carbamylation and increases the rate constants of the serial carbamylation and decarbamylation steps. Structural variation of the N-alkyl chain and of the aryl fused-ring system provides an accounting of bile salt modulation of the fatty acid and steroid binding sites, respectively. In that pseudo-substrate turnover of aryl carbamates proceeds by a three-step mechanism that is analogous to that for rapid turnover of lipid ester substrates, these investigations illuminate details of ligand recognition by the extended active site of cholesterol esterase that are prominent determinants of the substrate specificity and catalytic power of the enzyme. |
PubMedSearch : Feaster_1996_Biochemistry_35_16723 |
PubMedID: 8988009 |
Feaster SR, Lee K, Baker N, Hui DY, Quinn DM (1996)
Molecular recognition by cholesterol esterase of active site ligands: structure-reactivity effects for inhibition by aryl carbamates and subsequent carbamylenzyme turnover
Biochemistry
35 :16723
Feaster SR, Lee K, Baker N, Hui DY, Quinn DM (1996)
Biochemistry
35 :16723