Three-Dimensional Structure of Chymotrypsin Inactivated with (2S)-N-Acetyl-L-alanyl-L-phenylalanyl .alpha.-chloroethane: Implications for the Mechanism of Inactivation of Serine Proteases by Chloroketones

Abstract

The reaction of enantiomerically pure (2S)-N-acetyl-L-alanyl-L-phenylalanyl alpha-chloroethane with gamma-chymotrypsin was studied as a probe of the mechanism of inactivation of serine proteases by peptidyl chloroalkanes. It was determined crystallographically that the peptidyl chloroethane alkylates His57 with retention of configuration at the chiral center, indicating a double displacement mechanism. We think it likely that a Ser195-epoxy ether adduct is an intermediate on the inactivation pathway, although other possibilities have not been disproven. Kinetic data reported by others [Angliker et al. (1988) Biochem. J. 256, 481-486] indicate that the epoxy ether intermediate is not an irreversibly inactivated form of enzyme [a conclusion confirmed experimentally (Prorok et al. (1994) Biochemistry 33, 9784-9790)] and that both ring closure of the tetrahedral intermediate to form the epoxy ether and ring opening by His57 partially limit the first-order rate constant for inactivation, ki. The peptidyl chloroethyl derivative adopts a very different active site conformation from that assumed by serine proteases inactivated by peptidyl chloromethanes. Positioning the chloroethyl derivative into the conformation adopted by chloromethyl derivatives would cause the extra methyl group to make a bad van der Waals contact with the inactivator P2 carbonyl carbon, thereby preventing the formation of the invariant hydrogen bond between the inactivator P1 amide nitrogen and the carbonyl group of Ser214. We conclude that the unusual conformation displayed by the chloroethyl derivative is caused by steric hindrance between the extra methyl group and the rest of the inactivator chain.

A Mechanistic View of Enzyme Inhibition and Peptide Hydrolysis in the Active Site of the SARS-CoV 3C-like Peptidase

“…For example, chloromethyl ketone inhibitors usually form two covalent attachments each to one of the His57:Ser195 catalytic pair in their native conformation, possibly through a double displacement mechanism. 21 Interestingly, in the crystal structure of elastase complexed with the non-covalent inhibitor, trifluoroacetal-leucyl-alanyl-p-trifluoromethylphenylanilide (TFLA), the C β -O γ bond of Ser195 does undergo a similar rotation to avoid steric clashes with the trifluoro function of the inhibitor (PDB code 7EST). 22 This rotation in the side-chain of the catalytic serine/ cysteine residue not only increases slightly the distance between N ε2 of the assisting general base (His57 of elastase/His41 of 3CL pro ) and the nucleophilic atom, but also places the nucleophilic O γ /S γ atom in a position less coplanar with the imidazole ring of the histidine residue.…”

A Survey of Calpain Inhibitors

“…The mechanism of inactivation is however, different for the proteases. In concert with other cysteine proteases calpain inhibition by peptidyl chloromethyl ketones is due to alkylation of the active site thiolate [104] while inactivation of serine proteases involves alkylation of the active site histidine [105]. In attempts to generate selective protease inhibitors, peptidyl fluoromethyl ketones were investigated as enzyme inhibitors [106].…”

Haloacetamidine‐Based Inactivators of Protein Arginine Deiminase 4 (PAD4): Evidence that General Acid Catalysis Promotes Efficient Inactivation

“…[5] While this finding conclusively demonstrated the mode of inactivation-alkylation of the thiolate, the precise mechanism of inactivation has yet to be established.Although unclear, the mechanism of inactivation presumably proceeds through one of at least two routes: direct displacement of the halogen through an S N 2 mechanism or initial attack on the iminium carbon, followed by displacement of the halide to form a sulfonium ring, and ending with concomitant re-formation of the imine and opening of the sulfonium ring (Scheme 1). While the former possibility is the more intuitive, the latter is analogous to the mechanism by which the fluoromethylketones inactivate the cysteine proteases, [10][11][12] and therefore warranted further investigation.We began our investigations by examining the influence of pH on k inact /K I , that is, the second-order rate constant of enzyme inactivation. These studies were pursued because pH rate profiles can suggest the identity of catalytically important functional groups, in the inactivator or free enzyme, up to and including the first irreversible step of the reaction.…”