The stereospecificity and catalytic efficiency of the tryptophan synthase-catalysed exchange of the alpha-protons of amino acids

Abstract

13C-NMR has been used to follow the tryptophan synthase (EC 4.2.1.20) catalysed hydrogen-deuterium exchange of the pro-2R and pro-2S protons of [2-13C]glycine at pH 7.8. 1H-NMR has also been used to follow the tryptophan-synthase-catalysed hydrogen-deuterium exchange of the alpha-protons of a range of L- and D-amino acids at pH 7.8. The pK(a) values of the alpha-protons of these amino acids have been estimated and we have determined whether or not their exchange rates can be predicted from their pK(a) values. With the exception of tryptophan and norleucine, the stereospecificities of the first-order alpha-proton exchange rates are independent of the size and electronegativity of the amino acid R-group. Similar results are obtained with the second-order alpha-proton exchange rates, except that both L-tryptophan and L-serine have much higher stereospecificities than all the other amino acids studied.

Figure 1. ¹H-NMR spectra of L-serine before and after exchange of the α-proton with deuterium using α₂β₂ tryptophan synthase

The sample contained 0.05 M potassium phosphate buffer, pH 7.80, and 99.9 atom% ²H₂O. Spectrum A is of 0.02 M L-serine. Spectrum B is of 0.02 M L-[2-²H]serine produced by incubating L-serine with 7.68 μM wild-type α₂β₂ tryptophan synthase. Data acquisition for spectrum B commenced 7 h after the addition of tryptophan synthase. Both spectra resulted from 65 accumulations using a pulse width of 90°, an acquisition time of 2.56 s and an interpulse delay of 16 s.

Scheme 1. Minimal kinetic scheme for the tryptophan-synthase-catalysed exchange of the α-protons of amino acids

E, SH and SD are the free enzyme, the non-deuterated substrate and the deuterated substrate respectively. ESH and ESD are Schiff bases formed between pyridoxal 5′-phosphate and the α-amino group of the non-deuterated and deuterated amino acids respectively. EQ is a quinonoid intermediate resulting from the loss of either a proton from ESH or a deuteron from ESD.

Figure 2. The effects of amino acid concentration on the tryptophan-synthase-catalysed hydrogen–deuterium exchange rates of the α-protons of D-alanine and L-tryptophan at pH 7.80

(A) Concentrations of D-alanine of 0.01–0.4 M and tryptophan synthase active-site concentrations of 25.6–76.8 μM were used. (B) Concentrations of L-tryptophan of 0.0005–0.035 M and tryptophan synthase active-site concentrations of 0.08–2.4 μM were used. All samples contained 0.05 M potassium phosphate buffer, pH 7.80, and 99.9 atom% ²H₂O. The continuous lines in (A) and (B) were calculated using k_obs·[S]₀/[E]₀=k_a·[S]/([S]₀+K) and the fitted parameters: k_a=0.0211±0.0012 s⁻¹, K=0.0709±0.0117 M and k_a/K=0.297±0.052 M⁻¹·s⁻¹ (A); k_a=4.53±0.06 s⁻¹, K=0.000863±0.000068 M and k_a/K=5245±417 M⁻¹·s⁻¹ (B).

Figure 3. The effects of the length of the R-group on the first-order rate constant for (A) the serine hydroxymethyltransferase and (B) the tryptophan synthase catalysed exchange of the α-protons of L- and D-amino acids

Amino acids (number of carbons): glycine (1), alanine (2), aminobutyrate (3), norvaline (4), norleucine (5). (A) Serine hydroxymethyltransferase: data from Fitzpatrick and Malthouse [4]. (B) Tryptophan synthase: data from Table 1.