1. If the active site of a dipeptidase contains a glutamic acid residue (pKa 3.3) and a histidine residue (pKa 6.7), both of which must be charged for the substrate to bind, what is the optimal pH for substrate binding?
2. Lysozyme catalyzes the hydrolysis of C-O-C bonds between sugar residues in bacterial cell walls. The proposed catalytic mechanism for lysozyme requires that the side-chain group of aspartic acid-52 be in the ionized -COO- form and that of glutamic acid-35 be in the un-ionized -COOH form.
(a) What percentages of Asp and Glu side-chain groups would be in the ionized -COO- form at pH 5.0, the pH optimum for the hydrolysis of chitin by lysozyme, assuming pKas for the side-chains as given in Table 4.1 (attached) of Garrett and Grisham?
(b) In view of your answer to part (a), explain how it is possible for Glu-35 in lysozyme to be present in the un-ionized form.
3. A solution which contains a mixture of three tripeptides -- 1) Tyr-Arg-Ser, 2) Glu-Cys-Phe, and 3) Asp-Asp-His -- is chromatographed on CM-cellulose at three different pHs: 6.0 8.0, and 10.0. In what order will each of the peptides emerge from the column at each of these pHs? Use Table 4.1 in the Garrett and Grisham text for amino acid pKas. The pKa for the CM-cellulose is 4.90.
Please see the attached file.
Your questions are about the chemical properties of amino acids, and the ways in which these properties facilitate enzyme function (Qu. 1 and 2) and protein purification (Que 3).
1) Because the alpha-carboxyl and alpha-amino groups are involved in forming the peptide bond (the backbone of proteins), the functionality of amino acids within proteins is dependent upon the chemical nature of the side chain groups. The active site of an enzyme is a relatively small three-dimensional structure within the enzyme, to which substrates bind by multiple weak interactions (and sometimes covalent bonds). Polar residues are often found in active sites, to create a specific electrostatic environment within the cleft that forms the active site. Apparent pKa values for side chains of amino acids within proteins are often different from those for free amino acids.
You have been given the pKa values for a glutamic acid residue and a histidine residue within a dipeptidase. You are asked to determine the optimum pH for the enzyme based on the criterion that both of these residues must be charged for substrate binding to the active site. There are many examples of enzymes whose activity depends upon the acid/base properties of two key amino acid side chains within the active site, as in this example.
"Sensitivity to pH usually reflects an alteration in the ionization state of one or more residues involved in catalysis or occasionally in substrate binding. Plotting reaction velocity vs pH most often yields bell-shaped curve, where the inflection points approximate the pKa values of ionizable groups in the active site. At the pH optimum (midways between both pKa values) the greatest number of enzyme molecules are in the active form." (From http://www.langara.bc.ca/biology/mario/Biol2315notes/biol2315chap7.html)
The pKa value tells you about the acid-base properties of a side chain: deprotonation of acidic groups occurs at this pH. This makes sense if we look at the dissociation equation for the carboxylic acid portion of glutamic acid's side chain: deprotonation leads to a negative charge on this group.
Since this deprotonation occurs at pH = pKa = 3.3, we must be at this pH or above to have a charge on the glutamic acid side chain.
Similarly, in histidine we see a charge at pH values below the pKa 6.7, because deprotonation neutralizes a charge on the imidazole ring of the side chain at this pH:
Since we know that the optimum pH for this type of situation is halfway between the pKas for the two ionizable groups, we can easily ...
The solution provides answers to three questions involving pH substrate binding, ionization, and un-ionized forms.