Combined High-Resolution Neutron and X-ray Analysis of Inhibited Elastase Confirms the Active-Site Oxyanion Hole but Rules against a Low-Barrier Hydrogen Bond

Abstract

To help resolve long-standing questions regarding the catalytic activity of the serine proteases, the structure of porcine pancreatic elastase has been analyzed by high-resolution neutron and X-ray crystallography. To mimic the tetrahedral transition intermediate, a peptidic inhibitor was used. A single large crystal was used to collect room-temperature neutron data to 1.65 A resolution and X-ray data to 1.20 A resolution. Another crystal provided a low-temperature X-ray data set to 0.94 A resolution. The neutron data are to higher resolution than previously reported for a serine protease and the X-ray data are comparable with other studies. The neutron and X-ray data show that the hydrogen bond between His57 and Asp102 (chymotrypsin numbering) is 2.60 A in length and that the hydrogen-bonding hydrogen is 0.80-0.96 A from the histidine nitrogen. This is not consistent with a low-barrier hydrogen which is predicted to have the hydrogen midway between the donor and acceptor atom. The observed interaction between His57 and Asp102 is essentially a short but conventional hydrogen bond, sometimes described as a short ionic hydrogen bond. The neutron analysis also shows that the oxygen of the oxopropyl group of the inhibitor is present as an oxygen anion rather than a hydroxyl group, supporting the role of the "oxyanion hole" in stabilizing the tetrahedral intermediate in catalysis.

Cyclobutanone Analogues of β‐Lactam Antibiotics: β‐Lactamase Inhibitors with Untapped Potential?

“…Monitoring hemiketal formation in NMR experiments with [D 4 ]methanol, Dmitrienkoa nd co-workers noted that 78 (the exo cyclobutanone) was converted almost completely (98 %) into hemiketals 93 and 94,w hereas 79 (the endo analogue) gave much lower levels (15-40 %) of the corresponding hemiketals (95 and 96). [63,64] Inhibition of other serine proteases Broadening the scope of cyclobutanone b-lactam analogues beyondt ranspeptidase and b-lactamase inhibition, Reid and co-workerst ested their a-pepitamido monobactama nalogues 48 and 49 as inhibitors of the serine proteases human neutrophil elastase (HNE) and porcine pancreatic elastase (PPE). [27,28] Ta king this one step further,D mitrienko, Strynadka and coworkers succeeded in crystallising the 3a-methoxy analogue 70 in the active site of OXA-10, and thus were able to visualise the inhibitori na ction against as erine b-lactamase ( Figure 1).…”

Structural Basis for Human Monoglyceride Lipase Inhibition

“…46,47 The catalytic triad provides activation of the nucleophilic serine, which cleaves the ester bond of 2-AG when stabilized via its carbonyl group to the oxyanion hole formed by main-chain nitrogen atoms of Ala61 and Met133. The released glycerol molecule might diffuse toward a narrow "exit hole" located at the same level of the catalytic triad, while arachidonic acid could diffuse back to the top of the tunnel and exit the protein.…”

Pancreatic Elastases

“…The structures of many forms of free or inhibitor-bound pig PE I have been determined crystallographically to atomic resolution [14,27,41À43]. Recently, a combined high resolution neutron and X-ray diffraction analysis of porcine PE I-inhibitor complex revealed details of the molecular interactions around the oxyanion hole and of hydrogen bonding between residues of the catalytic triad, providing new insights into the catalytic mechanism of serine proteases [45]. The distance between His57 N (@1) and Asp102 O (@2) is 2.7 Å , indicating normal hydrogen bonding geometry [44].…”