1) Find kcat for a reaction in which Vmax is 8 x 10-3 mol/min and the reaction mixture contains two microgram of enzyme (the molecular weight of the enzyme is 100,000 D).
2) An enzyme is considered to have evolved to its most efficient form if
A) kcat is a large number
B) kcat/KM is near the diffusion-controlled limit
C) KM is a large number
D) kcat/KM is a very small number
E) KM is a small number
3) Determine the subunit composition of this protein from the following information:
Molecular mass of the native protein by gel filtration is 400 kD
Molecular mass by SDS-PAGE is 200 kD
Molecular mass by SDS-PAGE with 2-mercaptoethanol is 100 kD, 60 kD, and 40 kD
4) Treatment of a short peptide with trypsin gives four fragments with sequences:
Leu-Arg
Tyr-Thr-Ser-Val-Trp-Trp-Gly-Lys
Gln-Leu-Phe-Phe-Thr-Leu-Arg
Ala-Asp-Glu-Asn-Lys
Cleavage of the same peptide with chymotrypsin gives six fragments with the sequences:
Thr-Leu-Arg-Leu-Arg-Ala-Asp-Glu-Asn-Lys-Tyr
Thr-Ser-Val-Trp
Gln-Leu-Phe
Gly-Lys
Phe
Trp
What is the sequence of the intact peptide?
5) Treatment of a polypeptide with 2-mercaptoethanol yields two polypeptides:
(A) Val-Met-Cys-Arg-Gly-Gly-Phe-Arg-Cys-Leu-Ser
(B) Gly-Gln-Asp-Cys-Tyr-Val-Ile-Lys-Glu-Cys-Thr
Treatment with trypsin yields fragments with the following amino acid compositions:
Cys2, Glu, Leu, Ser, Thr
Arg, Gly2, Phe
Arg, Asp, Cys2, Gln, Gly, Ile, Lys, Met, Tyr, Val2
Using the two polypeptides above (A and B), indicate the positions of the disulfide bonds in the intact polypeptide.
Analysis of a peptide indicates that there is NO free animo terminal group. Digestion with trypsin yields three peptides with the sequences Ile-Cys-Met-Lys, Asp-Gln-Trp-Asp-Thr-Phe-Lys, and Ser-Gly-Tyr-Arg. Digestion with chymotrypsin yields three peptides with the sequences Arg-Asp-Gln-Trp, Asp-Thr-Phe, Lys-Ile-Cys-Met-Lys-Ser-Gly-Tyr. Draw a diagram of the original peptide.