Histidine titration

If the histidine titration curves are composite in the free protein as observed by Ruterjans and Witzel but not in the substrate complexes, the ks/Km curve should be distorted but not the ke curve. The opposite is observed. 

Kinetic studies indicated that external H interacted with the renal brush border Na /H exchanger (resistant-type) at a single site with apparent pKa 7.3-7.5. Because the imidazolium ring of histidine is the principal group that is titratable in this... 

Most frequently, protonation or deprotonation of protein occurs at ionizable, also called titratable, side chains, such as aspartate, glutamate or histidine. The ionization equilibrium of a titratable site,... 

Figure 10-4. The double- and single-site titration models for His and Asp groups [42]. (A) In the double site model, only one X is used for describing the equilibrium between the protonated and deprotonated forms, while the tautomer interversion process is represented by the variable x. (B) In the single-site model, protonation at different sites is represented by different X variables. HSP refers to the doubly protonated form of histidine. HSD and HSE refer to the singly protonated histidine with a proton on the h and e nitrogens, respectively. ASP1 and ASP2 refer to the protonated carboxylic acid with a proton on either of the carboxlate oxygens...

With multiple ionizable groups, such as in amino acids and proteins, each group titrates separately according to its pKa. The titration curves shown in Fig. 23-5 are for the amino acids glycine, histidine, and glutamate. 

Assignment of the isotropically shifted signals observed for the CuNiSOD example discussed in the previous paragraph has been achieved by means of anion titrations (not discussed here) and nuclear Overhauser enhancement spectroscopy (NOESY), to be discussed next. In Figure 3.24B the CuNiSOD active site is depicted with histidine nitrogens and protons identified for the discussion of the NOESY results. The copper(II) ion is coordinated to the N ligand atoms of his46... 

Among protein aromatic groups, histidyl residues are the most metal reactive, followed by tryptophan, tyrosine, and phenylalanine.1 Copper is the most reactive metal, followed in order by nickel, cobalt, and zinc. These interactions are typically strongest in the pH range of 7.5 to 8.5, coincident with the titration of histidine. Because histidine is essentially uncharged at alkaline pH, complex-ation makes affected proteins more electropositive. Because of the alkaline optima for these interactions, their effects are most often observed on anion exchangers, where complexed forms tend to be retained more weakly than native protein. The effect may be substantial or it may be small, but even small differences may erode resolution enough to limit the usefulness of an assay. 

The equilibrium dialysis experiment revealed that histidine-substituted salicylamide was selected as an RNA ligand. Subsequent binding analysis by UV titrations and Job plot revealed the histidine-substituted salicylamide Cu + complex bound the target RNA hairpin with an apparent dissociation constant of 150 nM. This binding constant likely reflects more complex binding processes than a simple 1 1 interaction, as the observed binding curve saturates well below the concentration of the histidine-substituted salicylamide, and thus the actual affinity of the complex for targeted RNA is probably lower. Importantly, however, titrations with the... 

In isoenzyme I, the titration behavior of zinc H20 is complicated due to the presence of three titratable active-site histidines as described in Section VI.D. Lindskog reports a value of 7.1 for the pKa of zinc-H20 (142), but higher values were obtained by other authors. The pH-rate profile for 4-nitrophenyl acetate esterase activity yields pKa = 7.45 (142). 

Similar isotope effects for human isoenzyme I (157c) on kc t and Km for C02 hydration are 1.7. Silverman and Tu (161) report an isotope effect of 2.5 for H2180 release and suggest that the intrinsic isotope effect of intramolecular H+ transfer might be significantly smaller in isoenzyme I than in isoenzyme II. Hence, the H20-splitting step might also limit the rate of C02 hydration in isoenzyme I. Human isoenzyme I has three titratable active-site histidines with pKa values... 

FIGURE 3-12 Titration curves for (a) glutamate and (b) histidine. The pKa of the R group is designated here as ptfR. 

E. Write the chemical reactions (including structures of reactants and products) that occur when the amino acid histidine is titrated with perchloric acid. (Histidine is a molecule with no net charge.) A solution containing 25.0 mL of 0.050 0 M histidine was titrated with 0.050 0 M HC104. Calculate the pH at the following values of Ve 0, 4.0, 12.5, 25.0, 26.0, and 50.0 mL. 

The approximation that histidine reacts completely with HC1 breaks down between 25 and 50 mL. If you used the titration equations in Table 11-6, you would find pH = 3.28, instead of 2.98, at Va = 26.0 mL. 

Figure 3-1 Titration curve for histidine. The solid line represents the uncorrected titration curve for 3 mM histidine monohydrochloride titrated with 0.2 M HCL to lower pH or with 0.2 M NaOH to higher pH assuming pKa values of 1.82, 6.00, and 9.17. The dashed line represents the corrected curve showing the number of protons bound or lost per mole of histidine monohydrochloride.

Using the pKa values from problem 3, construct the theoretical titration curve showing the equivalents of H+ or OH reacting with 1 mol of glycine as a function of pH. Note that the shape of this curve is independent of the pfCa. Sketch similar curves for glutamic acid (pK./s equal 2.19,4.25, and 9.67), histidine (pfCa s equal 1.82,6.00, and 9.17) and lysine (pfCa s equal 2.18,8.95, and 10.53). 

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