Abstract
Pre-protein sequence data was used to design substrates for SpsB, the bacterial signal peptidase I enzyme from Staphylococcus aureus. Key elements were an alkyl membrane anchor, proline at P5 and lysine at P2. The proline at P5 induced a helical turn in the lipopeptide, as deduced from NMR studies, from P6 to P2 in membrane mimetic solvents. The substrate Decanoyl-LTPTAKAASKIDD-OH was cleaved by SpsB, as expected, between the P1 and P1' alanines with a k(cat)/K(m) of 2.3x10(6) M(-1)s(-1) at pH 8.5. Insertion of proline at P1' converted substrates to competitive inhibitors, whilst the incorporation of an alpha-ketoamide at the cleavage site transformed substrates to time dependent inhibitors of SpsB.
MeSH terms
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Amides / chemistry
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Amides / pharmacology
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Amino Acid Sequence
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Binding Sites
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Dose-Response Relationship, Drug
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Enzyme Activation
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Escherichia coli / enzymology
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Escherichia coli / genetics
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Hydrogen-Ion Concentration
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Kinetics
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Lipoproteins / chemical synthesis*
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Lipoproteins / chemistry
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Lipoproteins / metabolism
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Magnetic Resonance Spectroscopy
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Membrane Proteins*
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Oligopeptides / chemical synthesis*
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Oligopeptides / chemistry
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Oligopeptides / metabolism
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Protein Conformation
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Recombinant Proteins / antagonists & inhibitors
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Recombinant Proteins / metabolism
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Sequence Homology, Amino Acid
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Serine Endopeptidases / drug effects
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Serine Endopeptidases / metabolism*
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Serine Proteinase Inhibitors / chemistry
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Serine Proteinase Inhibitors / pharmacology
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Staphylococcus aureus / enzymology*
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Staphylococcus aureus / genetics
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Substrate Specificity
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Time Factors
Substances
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Amides
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Lipoproteins
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Membrane Proteins
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Oligopeptides
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Recombinant Proteins
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Serine Proteinase Inhibitors
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Serine Endopeptidases
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type I signal peptidase