Binding Site Optimisation for Artificial Enzymes by Diffusion NMR of Small Molecules

Abstract

A binding site optimisation protocol for the design of artificial enzymes based on "small molecule-small molecule" binding studies by diffusion NMR is presented. Since the reaction chosen was the hydrolysis of ester 1 ([4-(4-carboxy-1-oxobutyl)-aminobenzyl]-phenethyl ester), an analogous phosphonate ester 2 ([4-(4-carboxy-1-oxobutyl)-aminobenzyl]-phosphonic phenethyl ester) was selected as a suitable transition state analogue (TSA). The key objective of the NMR studies was to find a unit with functional groups capable of binding to the acidic sites of the TSA. Nine dipeptides, mainly with basic and hydroxyl groups, were used and their affinity to the TSA was studied by measuring the change in the diffusion coefficient, D(pep), upon binding by pulse field gradient NMR. The value of D(pep) at 298 K in D(2)O at pD 5, 7 and 10 was measured both in free solution, and mixtures containing one dipeptide and the TSA. As both components are low molecular weight species with M < 500, a TSA-to-dipeptide ratio of 10:1 was used to detect significant changes in D(pep). The results revealed that dipeptides with basic residues show higher affinity to the TSA than those with hydroxyl or aliphatic side chains in aqueous solutions. The dipeptide showing the most significant relative change in D(pep) was H-Arg-Arg-OH, and the binding constant was estimated to be 86 L M(-1) by measuring D(pep) at varying concentrations of the TSA. In addition, binding of the TSA to a new water-soluble polymer with a polyallylamine backbone and randomly distributed Arg-Arg binding sites was examined, and the binding constant was estimated to be > or =1500 L M(-1). As confirmed by further catalytic activity tests, polymers containing Arg-Arg as a binding site are capable of significant rate accelerations in the hydrolysis of ester 1.

Motional timescale predictions by molecular dynamics simulations: Case study using proline and hydroxyproline sidechain dynamics

“…As higher concentrations may in principle lead to partial self-associations of peptides,87 we have used the experimental value of τ e = 29.7 ps at 298 K as a reference point for our MD simulations. From the analysis of τ e calculated for 14 parameter sets with a single non-zero V 3 term ( γ 3 = 0°, Table4), there is a linear correlation (Fig.…”

A Simple Protocol for the Modular Assembly of “Millipede” Artificial Enzymes

“…The translational diffusion coefficients of the nine dipeptides and the TSA 4 were measured in D 2 O at pD7 12. The value of D was measured both in free solution, and in mixtures containing one peptide and the TSA in a ratio of 3 m M :30 m M .…”

A Simple Protocol for the Modular Assembly of “Millipede” Artificial Enzymes

“…[*] As shown by our diffusion NMR measurements, [12] significant selfassociation effects are observed for the ester analogue 4 on increasing its concentration. Similar concentration-dependent selfassociation is also expected for ester 1, hence a dilute 0.5 mm solution of 3 was used in all our measurements and no variation of substrate concentration was attempted when comparing catalytic properties of polymers 5-8 in this study.…”

“…There was no evidence for amide hydrogen bond association. Even though it does not necessarily follow that the dipeptide with the strongest binding interaction with the TSA 4 will provide the most effective unit for catalysis, the ability to rank this affinity using the above NMR protocol [12] provides a valuable method for ordered selection.…”

“…[11] The translational diffusion coefficients of the nine dipeptides and the TSA 4 were measured in D 2 O at pD7. [12] The value of D was measured both in free solution, and in mixtures containing one peptide and the TSA in a ratio of 3 mm :30 mm. The diffusion coefficient of free TSA was measured to be 3.38 10 À10 m 2 s À1 .…”