The pepsin residue glycine-76 contributes to active-site loop flexibility and participates in catalysis

Abstract

Glycine residues are known to contribute to conformational flexibility of polypeptide chains, and have been found to contribute to flexibility of some loops associated with enzymic catalysis. A comparison of porcine pepsin in zymogen, mature and inhibited forms revealed that a loop (a flap), consisting of residues 71-80, located near the active site changed its position upon substrate binding. The loop residue, glycine-76, has been implicated in the catalytic process and thought to participate in a hydrogen-bond network aligning the substrate. This study investigated the role of glycine-76 using site-directed mutagenesis. Three mutants, G76A, G76V and G76S, were constructed to increase conformational restriction of a polypeptide chain. In addition, the serine mutant introduced a hydrogen-bonding potential at position 76 similar to that observed in human renin. All the mutants, regardless of amino acid size and polarity, had lower catalytic efficiency and activated more slowly than the wild-type enzyme. The slower activation process was associated directly with altered proteolytic activity. Consequently, it was proposed that a proteolytic cleavage represents a limiting step of the activation process. Lower catalytic efficiency of the mutants was explained as a decrease in the flap flexibility and, therefore, a different pattern of hydrogen bonds responsible for substrate alignment and flap conformation. The results demonstrated that flap flexibility is essential for efficient catalytic and activation processes.

The structure and function of Saccharomyces cerevisiae proteinase A

“…The highly conserved residue Gly76 likely enables this unusual conformation,19 since it adopts a positive torsion angle, φ, in PrA and bovine chymosin, whereas in other pepsin‐like aspartic proteinases Gly76 has a negative torsion angle. This proposal is consistent with work done in our laboratory which suggested that the role of Gly76 in porcine pepsin was the contribution of flexibility to the flap region 40…”

Confining a Protein-Containing Water Nanodroplet inside Silica Nanochannels

“…Indeed, the size and shape of the protein do not change significantly upon confining the pepsin-containing droplet in a mesopore, even at partial dehydration conditions, as can be also deduced from the data in Table 2, and from the superposition of the 0–20 ns configurations (Figure 5). On the whole, and in analogy with what observed for flat silica surfaces, these results suggest therefore that the pepsin catalytic properties [62,86] are maintained in a water nanodroplet confined in mesoporous silicas.…”

Flap flexibility amongst plasmepsins I, II, III, IV, and V: Sequence, structural, and molecular dynamics analyses

“…From the sequence analysis of the flap‐structure, it can be seen the PlmIV has a glycine residue situated at the flap tip (Gly78). The inherent flexibility of glycine accounts for the mobility and increased flexibility observed in PlmIV . Glycine is the smallest amino acid, due to it being achiral, with two hydrogen atoms composing the side chain.…”