Influence of sequence and charge on the specificity of lysyl oxidase toward protein and synthetic peptide substrates.
Lysyl oxidase initiates the covalent cross-linking of elastin and collagen by oxidizing lysine residues in these proteins to alpha-aminoadipic-delta-semialdehyde. Sequences surrounding susceptible lysines in elastin are considerably different from those in collagen and yet the same enzyme can oxidize both substrates. Possible bases of the specificity have been explored assaying for H2O2 release accompanying the oxidation of synthetic peptide and protein substrates. Rates of oxidation of random co-polymers were maximal with (Ala,Lys)n and decreased in the order (Val,Lys)n greater than (Leu,Lys)n greater than (Lys)n greater than (Phe,Lys)n greater than (Tyr,Lys)n. The ordered polymer (Ala-Lys-Glu)n was oxidized at only 3% of the rate of (Ala,Lys)n, implying inhibition by peptidyl glutamate. Consistent with this conclusion, kinetic analyses using ordered oligopeptides revealed that, relative to Ala-Ala-Lys-Ala-Ala, Km is increased 9.3-fold for lysine in Ala-Ala-Lys-Glu-Ala-Ala, 2.5-fold in Ala-Ala-Lys-Arg-Ala-Ala, and 1.8-fold in Ala-Ala-Glu-Lys-Ala-Ala. Tyrosine C-terminal to lysine in such peptides also increases Km 5-fold. In addition, lysyl oxidase oxidized lysine in various proteins with basic isoelectric points and was much less or not active against various acidic proteins. Lysyl oxidase was inactive against native bovine serum albumin but effectively oxidized albumin if albumin carboxyl functions were first amidated by chemical modification. These results suggest that peptides bind to lysyl oxidase in a preferred directional sense and indicate-that net anionic character as well as the specific position of anionic residues in substrates can selectively effect substrate potential. Implications of these results for the oxidation of elastin and collagen are discussed.
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D007526:Isoelectric Point; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D008249:Protein-Lysine 6-Oxidase / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D005971:Glutamates; D009842:Oligopeptides; D018698:Glutamic Acid; D000594:Amino Acid Oxidoreductases; D008249:Protein-Lysine 6-Oxidase; D008239:Lysine