Enhanced complexity and catalytic efficiency in the hydrolysis of phosphate diesters by rationally designed helix-loop-helix motifs.
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.
Razkin
Jesus
J
Department of Applied Chemistry, Public University of Navarra, 31006 Pamplona, Navarra, Spain. jrazk@unavarra.es
Lindgren
Johan
J
Nilsson
Helena
H
Baltzer
Lars
L
D000595:Amino Acid Sequence; D001665:Binding Sites; D040761:Biomimetic Materials / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D015195:Drug Design; D004952:Esters / Q000737:chemistry / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D006868:Hydrolysis / Q000187:drug effects; D057927:Hydrophobic and Hydrophilic Interactions; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D012492:Salts / Q000494:pharmacology
D004952:Esters; D010455:Peptides; D010710:Phosphates; D012492:Salts; D004247:DNA