The pH dependency of the kinetics of the CPA-catalyzed enolization of the ketonic substrate (R)-2-benzyl-3-(p-methoxybenzoyljpropionic acid ((-)-1) has been determined. The study of this relatively simple reaction allows us to examine the catalytic properties and ionization behavior of the enzyme-bound active-site bases and acids without the complications that would be encountered for peptides and esters where the formation and breakdown of a multiplicity of intermediates along the reaction pathway must be considered. The pKa values of 6.03 ± 0.35 and 6.04 ± 0.31 measured from the pH dependency of kcat for the Zn(II) and Co(II) CPA catalyzed enolization of (-)-1, respectively, correspond to the pka for the ionization of the γ-carboxyl of Glu-270. The binding of (-)-1 to CPA was investigated by examining the inhibition by this compound of the enzyme-catalyzed hydrolysis of O-(trans-p-chlorocinnamoyl)-L-β-phenyllactate. The KI-pH dependencies for the inhibitory activity of (-)-1 show that in alkaline solution one ionization of an enzyme-bound group occuss with pKa = 7.56 ±0.15 for the Zn(II) enzyme and pKa = 8.29 ± 0.32 for Co(II) CPA while the other occuss above pH 9. The pKa values in the vicinity of 8 represent the ionization of the phenolic hydroxyl of Tyr-248, in good agreement with earlier assignments of this pK. Finally, a reasonable interpretation of the pH dependency on a group with pKa <9 is that it corresponds to the ionization of the active-site metal ion bound water.
|Original language||English (US)|
|Number of pages||5|
|Journal||Journal of the American Chemical Society|
|State||Published - Jun 1983|
All Science Journal Classification (ASJC) codes
- Colloid and Surface Chemistry