MOLECULAR RECOGNITION BY CHOLESTEROL ESTERASE OF ACTIVE-SITE LIGANDS - STRUCTURE-REACTIVITY EFFECTS FOR INHIBITION BY ARYL CARBAMATES AND SUBSEQUENT CARBAMYLENZYME TURNOVER

Interactions of mammalian pancreatic cholesterol esterases from pig an d rat with a family of aryl carbamates CnH2n+1NHCOOAr [n = 4-9; Ar = p henyl, p-X-phenyl (X = acetamido, bromo, fluoro, nitro, trifluoromethy l), 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 carb amylation 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 inhibit ors allowed molecular recognition in the fatty acid binding and steroi d binding loci of the extended active site to be probed, and the elect ronic nature of the carbamylation transition state to be characterized . Optimal inhibitory activity is observed when the length of the carba myl function is rt = 6 and n = 7 for porcine and rat cholesterol ester ases, respectively, equivalent to eight- and nine-carbon fatty acyl ch ains. In contrast, inhibitory activity increases progressively as the partial molecular volume of the aromatic fragment increases. Hammett p lots for p-substituted phenyl-N-hexyl carbamates indicate that the rat e-determining step for carbamate inhibition is phenolate anion expulsi on. Effects of the bile salt activator taurocholate on the kinetically resolved phases of the pseudosubstrate turnover of aryl carbamates we re also studied. Taurocholate increases the affinity of the carbamate for the active site of cholesterol esterase in the reversible, noncova lent complex that precedes carbamylation and increases the rate consta nts of the serial carbamylation and decarbamylation steps. Structural variation of the N-alkyl chain and of the aryl fused-ring system provi des an accounting of bile salt modulation of the fatty acid and steroi d binding sites, respectively. In that pseudo-substrate turnover of ar yl carbamates proceeds by a three-step mechanism that is analogous to that for rapid turnover of lipid ester substrates, these investigation s illuminate details of ligand recognition by the extended active site of cholesterol esterase that are prominent determinants of the substr ate specificity and catalytic power of the enzyme.