Enhanced Complexity and Catalytic Efficiency in the Hydrolysis of Phosphate Diesters by Rationally Designed Helix‐Loop‐Helix Motifs

Abstract

HJ1, a 42-residue peptide that folds into a helix-loop-helix motif and dimerizes to form a four-helix bundle, successfully catalyzes the cleavage of "early stage" DNA model substrates in an aqueous solution at pH 7.0, with a rate enhancement in the hydrolysis of heptyl 4-nitrophenyl phosphate of over three orders of magnitude over that of the imidazole-catalyzed reaction, k(2)(HJ1)/k(2)(Im) = 3135. The second-order rate constant, k(2)(HJ1) was determined to be 1.58x10(-4) M(-1) s(-1). The catalyst successfully assembles residues that in a single elementary reaction step are capable of general-acid and general-base catalysis as well as transition state stabilization and proximity effects. The reactivity achieved with the HJ1 polypeptide, rationally designed to catalyze the hydrolysis of phosphodiesters, is based on two histidine residues flanked by four arginines and two adjacent tyrosine residues, all located on the surface of a helix-loop-helix motif. The introduction of Tyr residues close to the catalytic site improves efficiency, in the cleavage of activated aryl alkyl phosphates as well as less activated dialkyl phosphates. HJ1 is also effective in the cleavage of an RNA-mimic substrate, uridine-3'-2,2,2-trichloroethyl phosphate (leaving group pK(a) = 12.3) with a second-order rate constant of 8.23x10(-4) M(-1) s(-1) in aqueous solution at pH 7.0, some 500 times faster than the reaction catalyzed by imidazole, k(2)(HJ1)/k(2)(Im) = 496.

De novoprotein design, a retrospective

“…Subsequent rational improvement of the design allowed for introduction of enantioselective recognition of substrates (Broo et al ., 1998), a hallmark of natural proteins, and for elucidation of the role the p K a of the active residue as well as the geometry of the active site on catalysis (Broo et al ., 1998; Baltzer et al ., 1999). Expansion of the active site in the bundles to include additional residues to provide transition state stabilization allowed for hydrolysis of challenging phosphoester substrates, including uridine 3′−2,2,2-trichloroethylphosphate, a mimic of RNA (Razkin et al ., 2007, 2008). The simple architecture of KO-42 is nonetheless amenable to introduction of binding sites for complex substrates, whose recognition relies on multiple substrate–protein interactions.…”

Catalytic peptide assemblies

“…Further improvement of the catalytic activity was achieved by introducing tyrosine residues close to the active site. 42…”

Biocatalysts Based on Peptide and Peptide Conjugate Nanostructures

“…The catalytic activity was further enhanced by incorporation of tyrosine close to the active site based on two histidine residues flanked by four arginines and two adjacent tyrosine residues. 125 …”