Sulfhydryl groups ionization constants

We have previously reviewed some of the evidence for conformational changes induced by ligands, including the protection by substrate against dinitrophenylation of sulfhydryl groups and by both substrate and AMP against acetylation of specific tyrosine residues. The pK values for some tyrosine residues in the protein may be shifted by more than one pH unit on addition of the substrate (see above). The addition of AMP will also induce small changes in the ionization constants for the tyrosine residues with the lowest p.K values (40). 

It may seem odd that the technique of spectrophotometric titration, so extensively applied to the tyrosyl residues of proteins, has not been used in the study of protein sulfhydryl groups. The only study of this sort known to the author is the determination of the ionization constant of —SH grouj of thiolated gelatin (introduced by reaction with A -acetylhomocysteine thiolactone). By measuring the change in absorptivity as a function of pH at 2380 A, Benesch and Benesch (1958) found the apparent acidity of the thiol groups of the modified gelatin (pK = 9.8) to be nearly the same as that of an appropriate model compound, A -acetylhomocysteine (pK = 10.0). 

A minimal schematic outline of the proposed reaction steps for the hydrolysis of BAA is shown in Figure 28 (159). If it is assumed that the rate-limiting step governed by fci represents the formation of a thiol ester bond, reasonable explanations for the effect of pH and dielectric constant on fci are apparent. The presence of an essential, ionized /3-carboxyl group in the enzyme and of a cationic group in the most sensitive substrates suggests that an ionic interaction between enzyme and substrate (II in Fig. 28) enhances the rate of formation of ES. If the effect of pH on the alkaline side of the ki curve (Fig. 26) is due to titration of the sulfhydryl group, it is evident that the... 

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