Question

(7 pts) What is the sequence of the following HEXAPEPTIDE? *PROVIDE WORK/ANSWER for B-F! (Use deductive reasoning) Observations from Sequencing Experiments: A. The Sequence contains the following amino acids: Y, R, M, K, G, D B. Reaction with 1-fluoro-2,4-dinitrobenzene yielded DNP-G. ___ ___ ___ ____ ___ ___ y = 0.058x + 0.0081 y = 0.2711x + 0.0105 -0.02 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 Velocity ( mM/min) -1 [S] mM-1 Lineweaver-Burk No Inhibitor With Inhibitor Linear (No Inhibitor) Linear (With Inhibitor) 2 C. Treatment with chymotrypsin yielded two tripeptides. ___ ___ ___ ____ ___ ___ D. Treatment with trypsin yielded three dipeptides. ___ ___ ___ ____ ___ ___ E. Treatment with carboxypeptidase yielded methionine. ___ ___ ___ ____ ___ ___ F. N-terminal sequencing of one of the chymotrypsin-produced tripeptides yielded DNP-K ___ ___ ___ ____ ___ ___ Explanation and summary of protein sequencing techniques • 1-fluoro-2,4, dinitrobenzene (DNFB) is used to identify N-terminal amino acid of a peptide, and provides the following product: DNP-AA (where AA= the N-terminal amino acid of the peptide). • Trypsin is an enzyme that cleaves a peptide at the C-terminal side of R and K. • Chymotrypsin is an enzyme that cleaves a peptide at the C-terminal side of F, Y and W. • Carboxypeptidase is an enzyme that cleaves off the C-terminal amino acid of any peptide. THE HEXAPEPTIDE SEQUENCE: ???

Answer 1

G-R-Y-K-D-M

B). G is the N-terminal amino acid as the reaction with with 1-fluoro-2,4-dinitrobenzene yielded DNP-G. DNP treatment cleaves off the N-terminal amino acid.

C). Treatment with chymotrypsin yielded two tripeptides: G-R-Y and K-D-M.

Chymotrypsin cleaves a peptide at the C-terminal side of F, Y and W.

D). Treatment with trypsin yielded three dipeptides: G-R, Y-K, and D-M.

Trypsin cleaves a peptide at the C-terminal side of R and K.

E). M is the C-terminal amino acid as the treatment with carboxypeptidase yielded methionine.

F). N-terminal sequencing of one of the chymotrypsin-produced tripeptides yielded DNP-K.

One of the tripeptides produced by chymotrypsin treatment is K-D-M. In this, K is the N-terminal amino acid.