Dependence of transition-state structure on acyl chain length for cholesterol esterase-catalyzed hydrolysis of lipid p-nitrophenyl esters

Nucleophilic and general acid catalysis at physiological pH by a designed miniature esteraseElectronic supplementary information (ESI) available: NOESY and TOCSY spectra; Table with NMR assignments and observed NOEs; complete set of kinetic data for Art-Est catalysis; mass spectrum of fumaryl-Art-Est intermediate; rate constants for the spontaneous and catalysed ester hydrolysis. See http://www.rsc.org/suppdata/ob/b4/b404730c/. Coordinates for the structure of Art-Est have been deposited in the Brookhaven

“…A solvent kinetic isotope effect of this magnitude is generally interpreted as an indication of strong hydrogen bonding in the transition state and/or general acid base catalysis. The solvent KIE observed here, which was similar to the values typically observed for natural esterases, 18 suggested that Art-Est stabilised the transition state preceding the acyl intermediate through the protonated side-chain of His-18.…”

“…Art-Est followed a mechanism similar to that observed for natural esterases with active sites deeply buried within their structure, albeit with reduced catalytic activity. 18 The active site of Art-Est was based on two histidines with perturbed pK a values due to their proximity to one another and to neighbouring residues such as Glu-15, Arg-25 and Arg-26. This active site configuration led to a bell-shaped pH dependence of the catalytic rates.…”

Polymer Concentration-Controlled Substrate Specificity in Solvolysis of p-Nitrophenyl Alkanoates Catalyzed by 4-(Dialkylamino)pyridine-Functionalized Polymer in Aqueous Methanol Solution

“…Extensive studies have shown that the increase of hydrophobic effects of amphiphilic macromolecules, i.e., the increase in ratio of hydrophobic to hydrophilic components and the addition of salting-out agents, leads to changes of aggregate morphology from spheres to rods, and to vesicles in appropriate solvents. , 4-(Dialkylamino)pyridine-functionalized polymers have been regarded as useful and simple model systems for obtaining a better understanding of the origins of enzymic efficiency and selectivity. − Recently, we have reported ion-induced substrate specificity in solvolysis of p -nitrophenyl alkanoates 2 ( n = 2−18) catalyzed by polymer 1 containing the 4-(dialkylamino)pyridine functionality and a bis(trimethylene)disiloxane backbone (Scheme ) . The tris(hydroxymethyl)methylammonium ion as a salting-in ion induces the same substrate specificity for 2 ( n = 6) in aqueous Tris buffer solution that is obtained with cholesterol esterase for the same hydrolysis reaction . The addition of salting-out agent NaCl induces a substrate specificity change from 2 ( n = 14) to 2 ( n = 12) in 50:50 (v/v) methanol−aqueous phosphate buffer solution 7c…”

Control of Substrate Specificity in Polymer-Catalyzed Solvolysis Reactions of p-Nitrophenyl Alkanoates by Changing the Buffer System

“…Apparently, the substrate specificity in the 1 -catalyzed solvolysis of 2 ( n = 2−18) is controlled by changing the buffer system in buffered aqueous methanol solution. Although enzymes such as elastase, chymotrypsin, and cholesterol esterase and certain synthetic catalysts demonstrate the substrate preference in the solvolysis reactions of 2 , ,,, we are not aware of any catalytic systems that show the substrate specificity controlled by the nature of the buffer system.

1 Pseudo-first-order rate constants ( k obsd ) for the solvolysis of p -nitrophenyl alkanoates 2 ( n = 2−18, 5.0 × 10 -5 M) catalyzed by 1 as a function of alkanoate chain length ( n ) in 1:1 (v/v) methanol−aqueous Tris buffer (0.05 M Tris H + /Tris, pH 8.0) solution at 30 °C: (·) 7.5 × 10 -5 unit mol L -1 1 ; (▴) 2.5 × 10 -5 unit mol L -1 1 ; (▵) 5.0 × 10 -6 unit mol L -1 1 ; (▪) in the absence of 1 .

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“…4-(Dialkylamino)pyridine-functionalized polymers have been regarded as useful and simple model systems for obtaining a better understanding of the origins of enzymic efficiency and selectivity. − We have recently made an attempt to investigate such a model system to gain insight into the dominant control factors in solvolysis of p -nitrophenyl esters 2 ( n = 2−18) catalyzed by 4-(dialkylamino)pyridine-functionalized polymer 1 . − The mechanism of the reaction involves the attack by nucleophile 1 at the carbonyl group of substrates 2 and the formation of an N -acylpyridinium intermediate where the breakdown of the intermediate is the rate-determining step in the catalytic reactions . Strikingly, we have found ion-induced substrate specificity in the 1 -catalyzed solvolysis of 2 ( n = 2−18) in aqueous and methanol−water solutions. , Salting-in effects of the tris(hydroxymethyl)methylammonium ion in aqueous Tris buffer solution lead to the same substrate specificity for 2 ( n = 6) that is obtained with the enzyme, cholesterol esterase, for the same hydrolysis reaction …”