Organic Chemistry

Section 23.12 Determining the Primary Structure of a Protein 987

None of the exopeptidases or endopeptidases that we have mentioned will catalyze the

hydrolysis of an amide bond if proline is at the hydrolysis site. These enzymes recog-

nize the appropriate hydrolysis site by its shape and charge, and proline’s structure

causes the hydrolysis site to have an unrecognizable three-dimensional shape.

Cyanogen bromide causes the hydrolysis of the amide bond on the

C-side of a methionine residue. Cyanogen bromide is more specific than the en-

dopeptidases about what peptide bonds it cleaves, so it provides more reliable infor-

mation about the primary structure (the sequence of amino acids). Because cyanogen

bromide is not a protein and therefore does not recognize the substrate by its shape,

cyanogen bromide will still cleave the peptide bond if proline is at the cleavage site.

The first step in the mechanism for cleavage of a peptide bond by cyanogen bro-

mide is attack by the highly nucleophilic sulfur of methionine on cyanogen bromide.

Formation of a five-membered ring with departure of the weakly basic leaving group

is followed by acid-catalyzed hydrolysis, which cleaves the protein (Section 18.6).

Further hydrolysis can cause the lactone (a cyclic ester) to open to a carboxyl group

and an alcohol group (Section 17.11).

H 2 O

H 2 O

N

RR′

NHCHCNHCH H 3 NCHC

OO

NHCHCNHCH

R

CH 2

CH 2

OH

CH 2

CH 2

O

O

O O

COH C O

RR′

NHCHCNHCH

O CH 2 O

CH 2

C NHCHC+

+

+

+ CH 3 SC

HCl

HCl

R′ RR′

NHCHCNHCH

OOO

NHCHCNHCH NHCHC

R

CH 2

CH 2

S

CH 3

CH 2

CH 2

S

CH 3

O O

C

CN

CN

Br

O

C NHCHC

+

mechanism for the cleavage of a peptide bond by cyanogen bromide

+Br−

Ala-Lys-Phe-Gly-Lys-Trp-Ser-Arg-Met-Val-Arg-Tyr-Leu-His

cleavage by cyanogen bromide

(BrC‚N)

Ala-Lys-Pro Leu-Phe-Pro Pro-Phe-Val

trypsin will not cleave chymotrypsin will not cleave chymotrypsin will cleave

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