Formation and stability of the enolates of N-protonated proline methyl ester and proline zwitterion in aqueous solution: a nonenzymatic model for the first step in the racemization of proline catalyzed by proline racemase.

Rate constants for the hydrolysis of L-proline methyl ester to form proline and methanol in D(2)O buffered at neutral pD and 25 degrees C and the deuterium enrichment of the proline product determined by electrospray ionization mass spectrometry are reported. The data give k(DO) = 5.3 +/- 0.5 M(-1) s(-1) as the second-order rate constant for carbon deprotonation of N-protonated proline methyl ester by deuterioxide ion in D(2)O at 25 degrees C and I = 1.0 (KCl). The data provide good estimates of carbon acidities of pK(a) = 21 for N-protonated proline methyl ester and pK(a) = 29 for proline zwitterion in water and of the second-order rate constant k(HO) = 4.5 x 10(-5) M(-1) s(-1) for carbon deprotonation of proline zwitterion by hydroxide ion at 25 degrees C. There is no detectable acceleration of the deprotonation of N-protonated proline methyl ester by the Brønsted base 3-quinuclidinone in water, and it is not clear that such Brønsted catalysis would make a significant contribution to the rate acceleration for deprotonation of bound proline at proline racemase. A comparison of the first-order rate constants k(HO)[HO(-)] = 4.5 x 10(-11) s(-1) for deprotonation of free proline zwitterion in water at pH 8 and k(cat) = 2600 s(-1) for deprotonation of proline bound to the active site of proline racemase at pH 8 shows that the enzymatic rate acceleration for proline racemase is ca. 10(13)-fold. This corresponds to a 19 kcal/mol stabilization of the transition state for deprotonation of the enzyme-bound carbon acid substrate by interaction with the protein catalyst. It is suggested that (1) much of the rate acceleration of the enzymatic over the nonenzymatic reaction in water may result from transfer of the substrate proline zwitterion from the polar solvent water to a nonpolar enzyme active site and (2) the use of thiol anions rather than oxygen anions as Brønsted bases at this putative nonpolar enzyme active site may be favored, because of the smaller energetic price for desolvation of thiol anions than for desolvation of the more strongly solvated oxygen anions.