Bovine serum albumin-catalyzed deprotonation of [1-(13)C]glycolaldehyde: protein reactivity toward deprotonation of the alpha-hydroxy alpha-carbonyl carbon

Abstract

Bovine serum albumin (BSA) in D(2)O at 25 degrees C and pD 7.0 was found to catalyze the deuterium exchange reactions of [1-(13)C]glycolaldehyde ([1-(13)C]GA) to form [1-(13)C,2-(2)H]GA and [1-(13)C,2,2-di-(2)H]GA. The formation of [1-(13)C,2-(2)H]GA and [1-(13)C,2,2-di-(2)H]GA in a total yield of 51 +/- 3% was observed at early reaction times, and at later times, [1-(13)C,2-(2)H]GA was found to undergo BSA-catalyzed conversion to [1-(13)C,2,2-di-(2)H]GA. The overall second-order rate constant for these deuterium exchange reactions [(k(E))(P)] equals 0.25 M(-1) s(-1). By comparison, (k(E))(P) values of 0.04 M(-1) s(-1) [Go, M. K., Amyes, T. L., and Richard, J. P. (2009) Biochemistry 48, 5769-5778] and 0.06 M(-1) s(-1) [Go, M. K., Koudelka, A., Amyes, T. L., and Richard, J. P. (2010) Biochemistry 49, 5377-5389] have been determined for the wild-type- and K12G mutant TIM-catalyzed deuterium exchange reactions of [1-(13)C]GA, respectively, to form [1-(13)C,2,2-di-(2)H]GA. These data show that TIM and BSA exhibit a modest catalytic activity toward deprotonation of the alpha-hydroxy alpha-carbonyl carbon. We suggest that this activity is intrinsic to many globular proteins, and that it must be enhanced to demonstrate meaningful de novo design of protein catalysts of proton transfer at alpha-carbonyl carbon.

Figure 1

Portions of the ¹H NMR spectrum at 500 MHz of the reaction mixture obtained from the reaction of [1-¹³C]-GA (20 mM) for 120 h in the presence of 0.15 mM BSA in D₂O at pD 7.0, 25 °C and I = 0.10 (NaCl). A - The spectrum in the region of the C-2 hydron(s) of the isotopomers of GA hydrate; B - The spectrum in the region of the C-1 hydron of the isotopomers of GA hydrate.

Figure 2

Time course for the formation of products of the reaction of [1-¹³C]-GA (20 mM) in the presence of 0.15 mM BSA in D₂O at pD 7.0, 25 °C and I = 0.10 (NaCl), where f_P is the fractional yield of the product at the given reaction time. Key: (◆), fractional yield of [1-¹³C, 2,2-di-²H]-GA; (●), fractional yield of [1-¹³C, 2-²H]-GA.

Scheme 1

Scheme 2

Scheme 3

Chart 1