title: Peptidyldiazomethanes. A novel mechanism of interaction with prolyl endopeptidase.
abstract: Peptidyldiazomethanes with proline in the P1 position were found to be competitive slow-binding inhibitors of prolyl endopeptidase. Progress-curve experiments monitoring the increase in the degree of inhibition with time indicated that the kinetic mechanism involved an initial complex that isomerized to form a tighter complex. Reversibility of the inhibited complex was demonstrated by monitoring the regain of enzyme activity after removal of free inhibitor and dilution into an assay containing competing substrate. The kinetics of the reversal of inhibition indicated a more complicated inhibitory mechanism involving more than one pathway for reversal of the tight complex. A slow-binding mechanism of inhibition has not been previously observed with peptidyldiazomethanes. Incorporation of [3H]Ac-Ala-Ala-Pro-diazomethane into prolyl endopeptidase was observed after denaturation of the inhibited complex. The peptide labelled with [3H]Ac-Ala-Ala-Pro-diazomethane was isolated and found to contain the active-site serine residue.
authors.affiliation: Friedrich-Miescher-Institut, Basel, Switzerland.
authors.forename: S R
authors.initials: SR
authors.lastname: Stone
authors.forename: D
authors.initials: D
authors.lastname: Rennex
authors.forename: P
authors.initials: P
authors.lastname: Wikstrom
authors.forename: E
authors.initials: E
authors.lastname: Shaw
authors.forename: J
authors.initials: J
authors.lastname: Hofsteenge
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D003978:Diazomethane / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011392:Proline / Q000737:chemistry; D000085822:Prolyl Oligopeptidases; D011480:Protease Inhibitors / Q000494:pharmacology; D011489:Protein Denaturation; D012694:Serine; D012697:Serine Endopeptidases; D013552:Swine
chemical_list: D010455:Peptides; D011480:Protease Inhibitors; D012694:Serine; D003978:Diazomethane; D011392:Proline; D010450:Endopeptidases; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases