BL/CH 401 Lecture #5 - Protein Primary Structure

Cleavage of the Protein into Smaller Peptides

So after you have the N- and C-termini determined,

You cleave the protein into peptide fragments using:

1. Hydrolytic Enzymes (isolated from animal digestive system):

A. Trypsin cleaves at Lys and Arg

B. Chymotrypsin cleaves at Aromatic AAs (Phe, Tyr & Trp)

2. Specific chemicals like Cyanogen Bromide (CNBr) which cleaves at Met

Trypsin catalyzes peptide bond hydrolysis with water:

{*Figure 13*}

Important to note that the AA following the target Lys/Arg can not be Pro

Why does trypsin not catalyze breakage of Lys-Pro and Arg-Pro bond?

Remember that Pro has an unusual alpha-amino group and an unusually inflexible structure...

hence, Lys-Pro & Arg-Pro peptide bonds do not bind to trypsin"s catalytic site well!

Chymotrypsin catalyzes peptide bond hydrolysis with water just like trypsin:

{*Figure 14*}

Important to note that the AA following the target Phe/Tyr/Trp can not be Pro

Why does chymotrypsin not catalyze breakage of Phe-Pro, Tyr-Pro and Trp-Pro bond?

For same reason that trypsin does not (see discussion above)!

For trypsin and Chymotrypsin the number of peptides equals the number of target AAs plus one more.

For example:

If protein has 4 Lys and 3 Arg then get 8 peptides.

Four will have Lys at C-terminal, three will have Arg at C-terminal,

and the carboxyl-terminal peptide will not contain Lys or Arg.

Cyanogen Bromide (CNBr) cleaves at Met and...

converts the Met into another amino acid called Homoserine lactone (HSL).

If a protein contains 3 Met,

then 4 peptides will be found - 3 with C-terminal HSL and one containing no HSL.

{*Figure 15*} CNBr cleavage of peptide

©Wilbur H. Campbell, 1995, 1996; wcampbel@mtu.edu

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