Polyethylene Imine Derivatives (‘Synzymes') Accelerate Phosphate Transfer in the Absence of Metal

Abstract

The efficient integration of binding, catalysis, and multiple turnovers remains a challenge in building enzyme models. We report that systematic derivatization of polyethylene imine (PEI) with alkyl (C(2)-C(12)), benzyl, and guanidinium groups gives rise to catalysts ('synzymes') with rate accelerations (k(cat)/k(uncat)) of up to 10(4) for the intramolecular transesterification of 2-hydroxypropyl-p-nitrophenyl phosphate, HPNP, in the absence of metal. The synzymes exhibit saturation kinetics (K(M) approximately 250 microM, k(cat) approximately 0.5 min(-1)) and up to 2340 turnovers per polymer molecule. Catalysis can be specifically and competitively inhibited by anionic and hydrophobic small molecules. The efficacy of catalysis is determined by the PEI derivatization pattern. The derivatization reagents exert a synergistic effect, i.e., their combinations increase catalysis by more than the sum of each single modification. The pH-rate profile for k(cat)/K(M) is bell shaped with a maximum at pH 7.85 and can be explained as a combination of two effects that both have to be operative for optimal activity: K(M) increases at high pH due to deprotonation of PEI amines that bind the anionic substrate and kcat decreases as the availability of hydroxide decreases at low pH. Thus, catalysis is based on substrate binding by positively charged amine groups and the presence of hydroxide ion in active sites in an environment that is tuned for efficient catalysis. Inhibition studies suggest that the basis of catalysis and multiple turnovers is differential molecular recognition of the doubly negatively charged transition state (over singly charged ground state and product): this contributes a factor of at least 5-10-fold to catalysis and product release.

Combining Medium Effects and Cofactor Catalysis: Metal‐Coordinated Synzymes Accelerate Phosphate Transfer by 108

“…Following earlier work by Klotz and others,18–26 we have demonstrated in previous studies that environmental effects can also be exploited in water by using synthetic polymeric enzyme models (′synzymes′). Synzymes are based on a polyethylene imine (PEI) backbone that is functionalised combinatorially to create a microenvironment in which desolvation, dispersion interactions and specific medium effects support catalysis 27–29. Synzyme libraries have been shown to contain efficient catalysts with rate accelerations up to 10 7 (in k cat / k uncat ) or 10 8 (in ( k cat / K m )/ k 2 ) and multiple turnovers 27–29.…”

Arginine Coordination in Enzymatic Phosphoryl Transfer: Evaluation of the Effect of Arg166 Mutations in Escherichia coli Alkaline Phosphatase

“…The guanidinium group itself may be especially suited to binding phosphate because of its ability to form two simultaneous hydrogen-bonding interactions with phosphate oxygens (12-15). However, mutagenesis studies in several enzymes have found that loss of the arginine side chain yields dramatic reduction in catalysis without apparent changes in substrate affinity, raising the possibility of specificity of the arginine interactions for transition state charge and/or geometry (8-11, 16).…”

Reversible Electrochemical Modulation of a Catalytic Nanosystem

“…The low residual activity of non-metalated Au NP 1 presumably originates from the presence of protonated TACN-ligands, at neutral or acidic pH. [19] In this study, Cu 2+ and Cd 2+ metal ions were deliberately chosen for a number of reasons. Firstly, measurement of the catalytic activity as a function of the amount of Cu 2+ and Cd 2+ added to Au NP 1 showed that both metal ions activated the system for catalysis, reaching a maximum when a stoichiom- ], the rate acceleration was significantly higher for Au NP 1·Cu 2+ (9 times, red squares) than for Au NP 1·Cd 2+ (2.5 times, green squares) when compared to the background (Figure 1 b, dashed line).…”