Bioc460 2008, Dr. Ziegler, © Spring 2008 Peptide ionization practice problems, p. 1 Practice Problems on Peptide Ionization Properties (solutions on next 2 pages) From learning objectives for Lecture 4 (Peptides): Be able to estimate the approximate net charge on a short peptide at any given pH. This requires being given (e.g., on cover sheets of exams) or knowing the approximate pKa values of the ionizable groups in peptides and proteins (the single α-amino group and single α-carboxyl group on the peptide, and any ionizable R groups) as well as the chemistry/charge properties of those groups in their conjugate acid and conjugate base forms. Ionizable group in peptides and proteins Approximate ("generic") pKa in peptides & proteins (from Berg, Tymoczko & Stryer, Biochemistry, 6th ed., 2007) Ionizable group in peptides and proteins Approximate ("generic") pKa in peptides & proteins (from Berg, Tymoczko & Stryer, Biochemistry, 6th ed., 2007) α-carboxyl 3.1 thiol 8.3 side chain carboxyl 4.1 aromatic hydroxyl 10.9 imidazole 6.0 ε-amino 10.8 α-amino 8.0 guanidino 12.5 •To answer questions about peptide ionization properties, you need to know a) the pKa values of the 9 ionizable groups in peptides (and proteins), b) the structures of those ionizable groups (or in the case of arginine and histidine, at least approximate structures) c) the acid-base properties of the group, how the group ionizes: Is the conjugate acid (protonated form) + charged, in which case its conjugate base (the unprotonated form) is neutral, or is the conjugate acid neutral, in which case its conjugate base is – charged? •In any question about the charge properties of a peptide, focus just on the ionizable groups -- make notes on the sequence. If you need to write out shorthand structures of the groups, do that for practice while you're learning the structures and properties of the various groups, but on an exam, the more "shorthand" you can use in your analysis, the less time it will take you. Study questions/problems: 1. What would be the approximate net charge for each of the following peptides at pH 1? at pH 5? at pH 7? at pH 9? at pH 11.5? at pH 13? (You can consult the table on the cover of the 2005 or 2006 Exam 1 for pKa values.) [Why is this useful? You're not likely to be electrophoresing short peptides and needing to figure out toward which pole they would move, right? But thinking about net charges on peptides helps you to learn the charge properties of the ionizable groups in proteins, which are very important in enzyme catalytic mechanisms, binding interactions between biomolecules, etc.] Approx. net charge on whole peptide at each pH pH 1 pH 5 pH 7 pH 9 pH 11.5 pH 13 A. Phe-Glu-Asn-Cys-Arg B. Met-Tyr-Leu-Lys-Asp C. Cys-Arg-Gln-His-Lys-Trp These aren't that complicated -- an exam question might just ask for the net charge at one pH, but it's instructive to look for practice at what happens to the charge at a number of different pHs, so that's what we're doing here. You can also make up your own peptide sequences for practice; they're more interesting if they have some ionizable side chains, not just non-ionizable R groups, since such peptides would only have the α-amino and α-carboxyl groups for ionizable groups.) 2. Suppose an enzyme needed a cysteine thiol (sulfhydryl) group for its catalytic activity, and catalysis required the Cys thiol to act as a nucleophile at the beginning of the catalytic cycle. That would mean the Cys residue had to be in its UNPROTONATED (conjugate base) form. If that particular Cys residue in that protein had a pKa of 7.5, what fraction (per cent, or proportion) of the total enzyme molecules would have the Cys R group in its ACTIVE form at pH 7.0?Bioc460 2008, Dr. Ziegler, © Spring 2008 Peptide ionization practice problems, p. 2 SOLUTIONS 1.A. Phe-Glu-Asn-Cys-Arg List all ionizable groups (I myself make handwritten notes right on/above/below the sequence): “+” or “–“ indicates the predominant (“majority”) charge state (more than half in this charge state); if pH is less than 1 unit away from pKa, so group is not 90% in that state as it would be if the pH were a full pH unit above or below the pKa, the predominant charge at that pH is in parentheses: (+) or (–). Approx. net charge on individual GROUP at each pH ionizable functional group pH 1 pH 5 pH 7 pH 9 pH 11.5 pH 13 α-NH2 (pKa ~8) (N-terminal amino group) + + + 0 0 0 Glu R carboxyl (pKa ~4.1) 0 – – – – – Cys R-SH (pKa ~8.3) 0 0 0 (–) – – Arg R-guanidino (pKa ~12.5) + + + + + (0) α-COOH (pKa~3.1) (C-terminal carboxyl group) 0 – – – – – APPROX. NET CHARGE ON WHOLE PEPTIDE AT THIS pH (sum of individual groups' charges) +2 0 0 –2 –2 –3 1.B. Met-Tyr-Leu-Lys-Asp List ionizable groups. Approx. net charge on individual GROUP at each pH ionizable functional group pH 1 pH 5 pH 7 pH 9 pH 11.5 pH 13 α-NH2 (pKa ~8) (N-terminal amino group) + + + 0 0 0 Tyr R aromatic OH (pKa ~10.9) 0 0 0 0 (–) – Lys R-ε-NH2 (pKa ~10.8) + + + + (0) 0 Asp R-carboxyl (pKa ~4.1) 0 – – – – – α-COOH (pKa~3.1) (C-terminal carboxyl group) 0 – – – – – APPROX. NET CHARGE ON WHOLE PEPTIDE AT THIS pH (sum of individual groups' charges) +2 0 0 –1 –3 –3 1. C. Cys-Arg-Gln-His-Lys-Trp List ionizable groups. Approx. net charge on individual GROUP at each pH ionizable functional group pH 1 pH 5 pH 7 pH 9 pH 11.5 pH 13 α-NH2 (pKa ~8) (N-terminal amino group) + + + 0 0 0 Cys R-SH (pKa ~8.3) 0 0 0 (–) – – Arg R-guanidino (pKa ~12.5) + + + + + (0) His R-imidazole (pKa ~6) + + 0 0 0 0 Lys R-ε-NH2 (pKa ~10.8) + + + + (0) 0 α-COOH (pKa~3.1) (C-terminal carboxyl group) 0 – – – – – APPROX. NET CHARGE ON WHOLE PEPTIDE AT THIS pH (sum of individual groups' charges) +4 +3 +2 0 –1 –2Bioc460 2008, Dr. Ziegler, © Spring 2008 Peptide ionization practice problems, p. 3 2. Suppose an enzyme needed a cysteine thiol (sulfhydryl) group for its catalytic activity, and catalysis required the Cys thiol to act as a nucleophile at the beginning of the catalytic cycle. That would mean the Cys residue had to be in its UNPROTONATED (conjugate base) form. If that particular Cys residue in that protein had a pKa of 7.5, what fraction (per cent, or proportion) of the total enzyme molecules would have the Cys R group in its ACTIVE form at pH 7.0? Whenever a
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