elastase

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The atomic coordinates used in this script are derived from PDB entries 1hax, 1haz and 1hb0.

Reaction intermediates in proteolysis by elastase

In theory: The peptide to be cleaved is bound in the enzyme noncovalently in a groove near to the catalytic triad. As schematically shown below, in the first reaction step the hydroxyl of Ser¹⁹⁵ performs a nucleophilic attack on the substrate amide bond to form an ester. The carboxyterminal part of the substrate peptide is set free by this action. Now the aminoterminal part of the substrate is covalently bound to the enzyme. In the next step His⁵⁷ abstracts a proton from a water molecule, which then attaches to the ester carbon. This gives rise to the oxyanion intermediate.

The X-ray analysis experiment: The heptapeptide human beta-casomorphin-7 (BCM7) is a poor substrate for porcine pancreas elastase. Therefore it was possible to trap reaction intermediates at low temperatures and to analyze their structure. The ester stage was the first intermediate

to be seen, with four aminoterminal amino acids of BCM7 bound to the elastase. A close up view of the reaction center shows the ester bond

with the cabonyl oxygen in hydrogen bridge distance to the backbone nitrogens of Ser¹⁹⁵ and Gly¹⁹³

. The ester carbon is in perfect planar sp² hybridisation

. Besides the H-bonds to Ser¹⁹⁵ and Gly¹⁹³ in the oxyanion hole

the substrate is held by additional H-bonds to a strand in the enzyme

, enlarging an antiparallel beta-sheet with the substrate.

For the reaction to proceed, His⁵⁷

now depletes a water molecule

of one of it's protons so this oxygen's now free electron pair can attack the substrate. The resulting oxyanion intermediate structure

shows a change in electron hybridization for the ester carbon

from planar sp² to tetrahedal sp³ (use the mouse to turn the molecule!). There is some stabilization of the intermediate by distributing the charge of the three oxygen atoms among the oxyanion hole and on the other side

by hdrogen bonding via a water molecule to Thr⁴¹

. In this state the hydrogen bonds of BMC7 forming the beta-sheet with the enzyme are somewhat stretched compared to the ester intermediate

. This will facilitate the dissociation of the cleaved substrate from the enzyme once the bond to Ser¹⁹⁵ is broken. The change from the ester intermediate state to the tetrahedral state is mediated by a short rise of pH. With a more alkaline condition the reaction fast proceeds and in the next stage the enzyme is found without substrate

.
Now what about the third triad amino acid, Asp¹⁰²? It never gets into bonding distance to the substrate. The nevertheless important role of this residue is to maintain a hydrogen bond network (to the catalytic His⁵⁷ and the sheet-forming Ser²¹⁴) and set the electrostatic potential of the reaction center

.
An impression of the whole enzyme at work is given in a spacefilling

view.

RC Wilmouth et al, X-ray snapshots of serine protease catalysis reveal a tetrahedal intermediate, Nature Struct. Biol. 8 (2001) 689-694
G Katona et al, X-ray structure of a serine protease acyl-enzyme complex at 0.95-Å resolution, J. Biol. Chem. 277 (2002) 21962-21970