Multinuclear magnetic resonance studies on serine protease transition state analogues

Abstract

31P Nuclear Magnetic Resonance (NMR) studies were performed on mono- and diisopropylphosphoryl derivatives of alpha-chymotrypsin, trypsin, and subtilisin. Questions addressed included the pKa of the active center Asp...His...Ser triad in both species. While the pKa in the diisopropylphosphoryl derivatives is near 7.4 (found in this and other laboratories earlier) and reflects a nearly normal imidazolium titration curve, the apparent pKa in the monoisopropylphosphoryl enzymes (obtained by "aging" of the diisopropylphosphoryl derivatives and monitored by 31P NMR) is between 9.7 and 11.4 depending on the protease. This latter "titration" of the 31P NMR signal is reversible and presumably reflects the interaction of the imidazolium positive charge with the monoanionic phosphodiester. Of the two tetrahedral intermediates, the properties of the monoisopropylphosphoryl enzyme are probably more representative of the tetrahedral oxyanionic intermediate invoked during peptide hydrolysis. The same NMR technique was used to determine the action of PAM (pyridine-2-aldoxime methiodide, a known "antidote" for acetylcholinesterase inactivated by diisopropylfluorophosphate), on the inactivated enzymes. It was clear that the "antidote" could reverse the diisopropylphosphorylation but was ineffective on the monoisopropylphosphoryl ("aged") enzyme. 11B NMR studies were performed on phenylboronic (PBA) acid and 3,5-bis-trifluoromethylphenylboronic acid in the absence and presence of chymotrypsin and subtilisin. At 22 degrees C the former, but not the latter, compound was in fast exchange between the free and enzyme bound states. The relaxation parameters could be calculated for the bound PBA in chymotrypsin and the fluorinated analogue in subtilisin and clearly indicated that the boron nucleus was tetrahedral in the active centers, a good analogue for the tetrahedral oxyanionic intermediate.

Low Barrier Hydrogen Bond Is Absent in the Catalytic Triads in the Ground State but Is Present in a Transition-state Complex in the Prolyl Oligopeptidase Family of Serine Proteases

“…In the case of OpB, the boronate complex with the tetrahedral anionic character evidently induces a stronger hydrogen bond between the Asp and N ␦1 H at the active center. These results are consistent with previous reports on transition-state analogs that install a negative charge on the N ⑀2 side, such as complexes of peptide boronates (13)(14)(15)(16)28), peptidyltrifluoromethylketones (29 -30), as well as the monoisopropylphosphoryl Ser 195 derivatives of chymotrypsin, trypsin, and subtilisin (31), all of which uniformly raise the pK a (increase the basicity) of the active center histidine substantially. Similarly elevated pK a of the active center His in going to the transition state would be expected with the developing oxyanion, thereby reducing the ⌬pK a between the histidine and the serine, and with possible concomitant strengthening of the hydrogen bond between the Asp and N ␦1 H.…”

13C and 1H NMR Studies of Ionizations and Hydrogen Bonding in Chymotrypsin-Glyoxal Inhibitor Complexes

“…Similar increases in the basicity of the active site histidine have also been observed when peptidyl trifluoromethyl ketones (25,32) or peptidyl boronic acid inhibitors (36) form negatively charged tetrahedral adducts with chymotrypsin. The pK a of the active site histidine residue is also raised when subtilisin is covalently modified to give the monoisopropylphosphoryl enzyme (37). The pK a shifts of monoisopropylphosphoryl chymotrypsin and subtilisin could be predicted if a dielectric constant of ϳ4 was used (38).…”

Key active site residues in the inhibition of acetylcholinesterases by soman

“…This is because the proton transferring from the catalytic Ser residue to the catalytic His remains on the His as indicated by the increased pK of His in the adducts [6,7]. The evolutionary destination of the proton is to transfer to the leaving group in the natural substrate in the next step.…”

“…We have previously reported [l-5] on the molecular origin of irreversible inhibition of serine hydrolases, as portrayed in Scheme 1, being the complete impairment of the acid base catalytic apparatus of the enzymes. This is because the proton transferring from the catalytic Ser residue to the catalytic His remains on the His as indicated by the increased pK of His in the adducts [6,7]. The evolutionary destination of the proton is to transfer to the leaving group in the natural substrate in the next step.…”