13C- and 1H-NMR studies of oxyanion and tetrahedral intermediate stabilization by the serine proteinases: optimizing inhibitor warhead specificity and potency by studying the inhibition of the serine proteinases by peptide-derived chloromethane and glyoxal inhibitors

Abstract

Catalysis by the serine proteinases proceeds via a tetrahedral intermediate whose oxyanion is stabilized by hydrogen-bonding in the oxyanion hole. There have been extensive (13)C-NMR studies of oxyanion and tetrahedral intermediate stabilization in trypsin, subtilisin and chymotrypsin using substrate-derived chloromethane inhibitors. One of the limitations of these inhibitors is that they irreversibly alkylate the active-site histidine residue which results in the oxyanion not being in the optimal position in the oxyanion hole. Substrate-derived glyoxal inhibitors are reversible inhibitors which, if they form tetrahedral adducts in the same way as substrates form tetrahedral intermediates, will overcome this limitation. Therefore we have synthesized (13)C-enriched substrate-derived glyoxal inhibitors which have allowed us to use (13)C-NMR and (1)H-NMR to determine how they interact with proteinases. It is hoped that these studies will help in the design of specific and highly potent warheads for serine proteinase inhibitors.

Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate–peptide complex structures

“…The currently used rhomboid inhibitors, isocoumarins, phosphonofluoridates and monocyclic β‐lactams (Vinothkumar et al , , ; Pierrat et al , ; Xue & Ha, ; Xue et al , ), were unsuitable as warheads because the stereochemical similarity of peptidyl conjugates of isocoumarins and β‐lactams to the acyl enzyme intermediate would be limited, and phosphonofluoridates have proven difficult to synthesise in the desired sequence diversity. We therefore turned our attention to peptidyl‐chloromethylketones (CMKs) (Fig A), whose complexes with serine proteases resemble the tetrahedral transition state intermediate (Mac Sweeney et al , ; Malthouse, ) and which are readily synthesisable. The commercially available CMKs TLCK (N‐α‐tosyl‐L‐lysine chloromethylketone) and TPCK (N‐α‐tosyl‐L‐phenylalanine chloromethylketone) had shown only weak inhibition of YqgP and Drosophila rhomboid 1 (Urban et al , ; Urban & Wolfe, ), but we reasoned that this could have been due to their unsuitable P1 residues (Lys or Phe), since P1 residues with large side chains are not tolerated in substrates by several rhomboids including GlpG (Strisovsky et al , ; Vinothkumar et al , ).…”

Steady-State Kinetics and Mechanism of LpxD, the N-Acyltransferase of Lipid A Biosynthesis

“…7) (14). Furthermore, because most oxyanion "holes" in serine proteases (44), protein N-myristoyl transferases (45) and lipases (46) feature backbone NH groups, we favor the alternative mechanistic scenario, shown in Scheme 2 and Fig. 8.…”

Oxyanion and tetrahedral intermediate stabilisation by subtilisin: Detection of a new tetrahedral adduct

“…X-ray crystallographic studies had led to the suggestion that the oxyanion of the tetrahedral intermediate might be stabilised by hydrogen bonding to the backbone amide groups of serine-195 and glycine-193 [1,2]. Subsequently there has been considerable interest in determining how the oxyanion is stabilised [1][2][3][4][5][6][7][8][9][10][11].…”