Slow tight-binding inhibition of prolyl endopeptidase by benzyloxycarbonyl-prolyl-prolinal.
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ABSTRACT:
SUBMITTER: Bakker AV
PROVIDER: S-EPMC1149592 | biostudies-other | 1990 Oct
REPOSITORIES: biostudies-other
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Project description:Inhibitors with dissociation constants in the micromolar to nanomolar range are important, but hard to characterize kinetically, especially when the substrate concentration in the assay is less than Km. When inhibition increases during the course of the assay (slow-binding inhibition) the concentration of substrate may decrease appreciably. Methods that take substrate depletion into account are described for analysing experiments in which the initial substrate concentration is below Km. Fitting progress curves gives the rate constants for the second (slow) step in a two-step mechanism. An approximate value for the overall dissociation constant may be determined from measurements of rates when the reaction is treated as a first-order process. When the concentrations of inhibitor and enzyme are comparable numerical methods are required. Procedures, suitable for implementation on a microcomputer, for the solution of the differential equations and the fitting of progress curves are described.
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Project description:Prolyl peptidases cleave proteins at proline residues and are of importance for cancer, neurological function, and type II diabetes. Prolyl endopeptidase (PEP) cleaves neuropeptides and is a drug target for neuropsychiatric diseases such as post-traumatic stress disorder, depression, and schizophrenia. Previous structural analyses showing little differences between native and substrate-bound structures have suggested a lock-and-key catalytic mechanism. We now directly demonstrate from seven structures of Aeromonus punctata PEP that the mechanism is instead induced fit: the native enzyme exists in a conformationally flexible opened state with a large interdomain opening between the beta-propeller and alpha/beta-hydrolase domains; addition of substrate to preformed native crystals induces a large scale conformational change into a closed state with induced-fit adjustments of the active site, and inhibition of this conformational change prevents substrate binding. Absolute sequence conservation among 28 orthologs of residues at the active site and critical residues at the interdomain interface indicates that this mechanism is conserved in all PEPs. This finding has immediate implications for the use of conformationally targeted drug design to improve specificity of inhibition against this family of proline-specific serine proteases.
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Project description:Prolyl endopeptidase (Prep) is a member of the prolyl peptidase family and is of interest because of its unique biochemistry and connections to cognitive function. Using an unbiased mass spectrometry (MS)-based peptidomics platform, we identified Prep-regulated peptides in the central nervous system (CNS) of mice by measuring changes in the peptidome as a function of Prep activity. This approach was validated by the identification of known Prep substrates, such as the neuropeptide substance P and thymosin-beta4, the precursor to the bioactive peptide Ac-SDKP. In addition to these known substrates, we also discovered that Prep regulates many additional peptides, including additional bioactive peptides and proline rich peptides (PRPs). Biochemical experiments confirmed that some of these Prep-regulated peptides are indeed substrates of the enzyme. Moreover, these experiments also supported the known preference of Prep for shorter peptides while revealing a previously unknown cleavage site specificity of Prep when processing certain multi-proline-containing peptides, including PRPs. The discovery of Prep-regulated peptides implicates Prep in new biological pathways and provides insights into the biochemistry of this enzyme.
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Project description:Maternal genotype has lifetime effects on progeny, but few specific genes, and no proteases, are known to underlie maternal effects. Prolyl endopeptidase (PREP) is a serine protease with putative substrates that regulate appetite or milk production.To test effects of PREP on obesity phenotypes in mice.Mice with a gene trap (GT) of PREP (PREP(gt/gt)) on the C57BL/6J (B6) background were generated. Minimal PREP protein was detected by western blot. In Experiment 1, direct effects of PREP were measured in littermate mice derived from intercrosses of heterozygotes (PREP(WT/gt)). In Experiment 2, maternal effects of PREP were measured in reciprocal crosses of heterozygous (PREP(WT/gt)) and wild-type (WT) (PREP(WT/WT)) males and females. DIETS: Mice were fed either low-fat (LF, Experiments 1 and 2) or high-fat (HF, Experiment 1) defined diets.Adiposity index (AI) was calculated from body weight (BW) and weights of four fat depots measured in 120-day-old mice. Fasting plasma glucose, insulin and leptin were measured. In vivo plasma alpha-MSH levels were measured by targeted quantitative peptidomics.Experiment 1-In intercross mice, there were significant diet effects, but few genotype effects. There were no genotype effects on BW or AI in males or females on either diet. Experiment 2-In contrast, reciprocal crosses of heterozygous males or females with WT B6 revealed highly significant parent of origin effects on all traits except body length. Progeny (WT and heterozygous genotypes and both sexes) born to female PREP(WT/gt) heterozygotes had fat pads that weighed as much as -twofold more at 120 days old than progeny born to male heterozygotes.Heterozygosity for PREP GT results in highly significant maternal effects, whereas homozygosity for the PREP(gt/gt) mutation has a much more limited direct effect.
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