Serine and Cysteine Peptidases: So Similar, Yet Different. How the Active-Site Electrostatics Facilitates Different...

The catalytic mechanisms of serine and cysteine peptidases are similar: the proton of the nucleophile (serine or cysteine) is transferred to the catalytic histidine, and the nucleophile attacks the substrate for cleavage. However, they differ in an important aspect: cysteine peptidases form a stable ion-pair intermediate in a stepwise mechanism, while serine peptidases follow a concerted mechanism. While it is known that a positive electrostatic potential at the active site of cysteine peptidases stabilizes the cysteine anion in the ion-pair state, the physical basis of the concerted mechanism of serine peptidases is poorly understood. In this work, we use continuum electrostatic analysis and quantum mechanical/molecular mechanical (QM/MM) simulations to demonstrate that a destabilization of an anionic serine by a negative electrostatic potential in combination with a compact active site geometry facilitates a concerted mechanism in serine peptidases. Moreover, we show that an anionic serine would destabilize the protein significantly compared to an anionic cysteine in cysteine peptidases, which underlines the necessity of a concerted mechanism for serine peptidases. On the basis of our calculations on an inactive serine mutant of a natural cysteine peptidase, we show that the energy barrier for the catalytic mechanism can be substantially decreased by introducing a negative electrostatic potential and by reducing the relevant distances indicating that these parameters are essential for the activity of serine peptidases. Our work demonstrates that the concerted mechanism of serine peptidases represents an evolutionary innovative way to perform catalysis without the energetically expensive need to stabilize the anionic serine. In contrast in cysteine peptidases, the anionic cysteine is energetically easily accessible and it is a very efficient nucleophile, making these peptidases mechanistically simple. However, a cysteine is highly oxygen sensitive, which is problematic in an aerobic environment. On the basis of the analysis in this work, we suggest that serine peptidases represent an oxygen-insensitive alternative to cysteine peptidases.