Interaction between bovine trypsin and a synthetic peptide containing 28 residues of the bait region of human alpha 2-macroglobulin.
The time course of the interaction between trypsin and a synthetic peptide corresponding to a segment (residues 676-703) of the bait region (residues 666-706) of human alpha 2-macroglobulin (alpha 2M) was studied by measuring the generation of cleavage products as a function of time by HPLC. Three primary cleavage sites for trypsin were present in the synthetic peptide. The fastest cleavage occurred at the bond corresponding to Arg696-Leu in alpha 2M with an estimated kcat/Km = 1-2 x 10(6) M-1.s-1. This value is of the same magnitude as that characterizing the interaction of alpha 2M and trypsin when taking into account the fact that alpha 2M is a tetramer, kcat/Km = 5 x 10(6) M-1.s-1 [Christensen, U. & Sottrup-Jensen, L. (1984) Biochemistry 23, 6619-6626]. The values of kcat/Km for cleavage at bonds corresponding to Arg681-Val and Arg692-Gly in alpha 2M were 1.5 x 10(5) M-1.s-1 and 1.3 x 10(5) M-1.s-1, respectively. Cleavage of intermediate product peptides was slower, with kcat/Km in the range 13-1.3 x 10(6) M-1.s-1. The value of Km determined for fast cleavage in the synthetic peptide was 8-10 microM. 1H-NMR spectroscopy indicated no ordered structure of the peptide. Hence, the very fast cleavage of the peptide is compatible with a loose structure that readily adopts a conformation favorable for recognition and cleavage by trypsin.
Thomsen
N K
NK
Department of Molecular Biology, University of Arhus, Denmark.
Christensen
U
U
Madsen
K
K
Sottrup-Jensen
L
L
D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000378:metabolism*
D010446:Peptide Fragments; D000511:alpha-Macroglobulins; D014357:Trypsin