固相合成による甘味タンパク質モネリンの構造活性相関

Abstract

Monellin, an intensely sweet protein, was isolated from the fruit of the West African plant, Dioscoreophyllum cumminsii Monellin consists of two noncovalently associated polypeptide chains, the A chain with 44 amino acid residues and the B chain with 50 residues. The tertiary structure has been determined, and the native conformation is essential for the sweet taste. Recently, the primary structure of monellin was unambiguously determined, and monellin was synthesized by the Fmoc solid-phase method. In an attempt to delineate the active site(s) of monellin, amino acid residues within potential active sites were replaced. Replacement of CysB41 by Ser led to a small change in sweetness. Replacement of IleB6, AspB7 or IleB8 by different amino acids resulted in a complete or marked loss of sweetness. Replacement of AspA16 or TyrA13 by other amino acids did not reduce the substantial sweetness. Replacement of each Lys residue by L-2-aminohexanoic acid (L-norleucine) significantly diminished the sweetness potency, but did not completely remove the sweetness. Analysis of the synthetic analogs by circular dichroism (CD) showed that the major structural features of monellin were not significantly altered. These findings suggest that IleB6, AspB7 and IleB8 are responsible for eliciting a sweet taste, and that one or more basic residues may also be important for eliciting a sweet taste. In the course of the investigation, the D-enantiomer of monellin was synthesized by the Fmoc solid-phase method, and crystallized by the hanging drop vapor diffusion method. The D-isomer was shown by CD to be the mirror image of the synthetic L-monellin, and was devoid of any sweetness (essentially tasteless). DL-Monellin was crystallized from a 1 : 1 mixture of the synthetic L- and D-monellin, and its CD spectrum did not show any absorption. DL-Monellin was 2000 times sweeter than sucrose on a weight basis (cf., monellin is 4000 times sweeter than sucrose) These results indicate that the crystals consist of the racemates of DL-monellin.