Inactivation of prolyl endopeptidase by a peptidylchloromethane. Kinetics of inactivation and identification of sites of modification.
The kinetics of inactivation of prolyl endopeptidase by acetyl-Ala-Ala-Pro-CH2Cl were studied by progress-curve methods in the presence of substrate. The kinetic mechanism was found to involve the formation of an initial complex between the enzyme and the chloromethane followed by an inactivation step. The substrate was shown to compete for the formation of the initial complex, indicating that binding at the active site was a prerequisite for inactivation. After reaction of the enzyme with [3H]acetyl-Ala-Ala-Pro-CH2Cl, it was possible to isolate five labelled peptides. Four of these peptides contained a cysteine residue as the site of modification, whereas the fifth peptide contained no cysteine and a histidine residue was identified as the site of modification. This residue (His-680) probably represents the active-site histidine of prolyl endopeptidase.
Stone
S R
SR
Friedrich Miescher-Institut, Basel, Switzerland.
Rennex
D
D
Wikstrom
P
P
Shaw
E
E
Hofsteenge
J
J
D000478:Alkylation; D000595:Amino Acid Sequence; D001665:Binding Sites; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology*; D000085822:Prolyl Oligopeptidases; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*
D009842:Oligopeptides; D015842:Serine Proteinase Inhibitors; D006639:Histidine; D010450:Endopeptidases; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases