Enzyme-specific activation versus leaving group ability.
Bottom Line: Enzyme-specific activation and the substrate mimetics strategy are effective ways to circumvent the limited substrate recognition often encountered in protease-catalyzed peptide synthesis.A key structural element in both approaches is the guanidinophenyl (OGp) ester, which enables important interactions for affinity and recognition by the enzyme--at least, this is usually the explanation given for its successful application.In this study we show that leaving group ability is of equal or even greater importance.
Affiliation: Institute for Molecules and Materials, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.Show MeSH
Mentions: We set out to locate the tetrahedral intermediate for the OGp compound. After exhaustive partial optimizations (in which the C–O bond connecting OGp to the central carbon of the tetrahedral intermediate was fixed), we came to the conclusion that nowhere along this internal coordinate did a stationary point exist. No optimum or saddle point could be found. Removing the restraint of the fixed C–O bond always resulted in dissociation upon optimization. A stable intermediate was found, however, when a hydrogen atom was added to the oxygen carrying the negative charge. This is in line with the commonly accepted hypothesis that, in the natural protein environment, hydrogen bonding in the oxyanion hole stabilizes the tetrahedral intermediate. As an equivalent amount of atoms and charge was required throughout the calculations, a hydrogen atom was added to the leaving group too. This corresponds with the accepted mechanism, in which the leaving group is protonated upon formation of the first tetrahedral intermediate. With the stable intermediates identified, the energy diagram (Figure 4) and the data (Table 2) were produced.
Affiliation: Institute for Molecules and Materials, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.