Peptidic phosphonylating agents as irreversible inhibitors of serine proteases and models of the tetrahedral intermediates.
Peptide analogues incorporating an electrophilic phosphorus moiety (2-6) have been synthesized and studied as inhibitors of a variety of serine proteases. Inhibition is irreversible and, for alpha-lytic protease (ALP), shown to result from covalent binding to the active site serine hydroxyl [Bone, R., Sampson, N. S., Bartlett, P.A., & Agard, D. A. (1991) Biochemistry (following paper in this issue)]. For reaction of human leukocyte elastase (HLE) with the thiophenyl esters 6s-V (Boc-AAPV psi [P = O(SPh)O]AA-OMe), 4s-V (BocAAPV psi [P = O(SPh)O]-Me), and 3s-V (Boc-V psi [P = O(SPh)O]AA-OMe), evidence is presented to suggest that the S4-S1 subsites, but not the S1' and S2' positions, are occupied by the inhibitors during the inactivation process. The selectivity that is observed between the proteases and the hexapeptide phosphonates 6o-V (Boc-AAPV psi [P = O(OPh)O]AA-OMe) and 6o-F (Boc-AAPF psi [P = O(OPh)O]AA-OMe) parallels that between these enzymes and their substrates: ALP and HLE are selectively inactivated by the ValP-containing analogue 6o-V, while subtilisin (SUB) shows a preference for the PheP derivative 6o-F. A detailed kinetic analysis of the enzyme-inhibitor interactions was complicated by the susceptibility of the inhibitors to enzymatic degradation. The configuration at phosphorus was found not to have a significant influence on the rate at which the inhibitors react with the peptidases. Moreover, in the case of inactivation of ALP by the hexapeptide 6o-V, the same covalent adduct is formed from both stereoisomers (Bone et al., 1991), indicating that one of these diastereomers undergoes substitution with retention of configuration.
Sampson
N S
NS
Department of Chemistry, University of California, Berkeley 94720.
Bartlett
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000494:pharmacology; D063065:Organophosphonates / Q000494:pharmacology*; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
D007202:Indicators and Reagents; D009842:Oligopeptides; D063065:Organophosphonates; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases