13C NMR study of how the oxyanion pKa values of subtilisin and chymotrypsin tetrahedral adducts are affected by different amino acid residues binding in enzyme subsites S1-S4.

A range of substrate-derived chloromethane inhibitors have been synthesized which have one to four amino acid residues. These have been used to inhibit both subtilisin and chymotrypsin. Using 13C NMR, we have shown that all except one of these inhibitors forms a tetrahedral adduct with chymotrypsin, subtilisin, and trypsin. From the pH-dependent changes in the chemical shift of the hemiketal carbon of the tetrahedral adduct, we are able to determine the oxyanion pKa in the different inhibitor derivatives. Our results suggest that in both the subtilisin and chymotrypsin chloromethane derivatives the oxyanion pKa is largely determined by the type of amino acid residue occupying the S1, subsite while binding in the S2-S4 subsites only has minor effects on oxyanion pKa values. Using free energy relationships, we determine that the different R groups of the amino acid residues binding in the S1 subsite only have minor effects on the oxyanion pKa values. We propose that the lower polarity of the chymotrypsin active site relative to that of the subtilisin active site explains why the oxyanion pKa is higher and more sensitive to the type of chloromethane inhibitor used in the chymotrypsin derivatives than in the subtilisin derivatives.

Enzymatic synthesis of isotopically labelled serine and tryptophan for application in peptide synthesis.

L-[1.2-13C2, 15N]Serine was prepared from [1,2-13C2, 15N]glycine on a gram scale by the use of the enzyme serine hydroxymethyltransferase. The reaction was monitored by 13C-NMR spectroscopy. This is the first simultaneously 13C- and 15N-labelled serine isotopomer so far reported. Part of the product was directly converted by tryptophan synthase to L-[1,2-13C2, 15N]tryptophan which could conveniently be purified and isolated as Boc-derivative in a yield of 71%. Most of the serine was isolated similarly but to remove remaining starting material in this case purification by column chromatography was required.

The effect of different amino acid side chains on the stereospecificity and catalytic efficiency of the tryptophan synthase-catalysed exchange of the alpha-protons of amino acids.

1H-NMR has been used to follow the tryptophan synthase (EC 4.2.1.20) c catalysed hydrogen-deuterium exchange of the alpha-protons of L- and D-alanine and -tryptophan. The first-order and second-order rate constants for exchange have been determined at pH 7.8 in the presence and absence of the allosteric effector, DL-alpha-glycerol 3-phosphate. In the presence of DL-alpha-glycerol 3-phosphate the stereospecificity of the tryptophan synthase-catalyzed first-order exchange rates was in the order tryptophan > alanine > glycine. This increase in stereospecificity was largely due to the decrease in the magnitude of the first-order exchange rate of the slowly exchanged alpha-proton. A similar increase in the stereospecificity of the second-order exchange rates for alanine was also largely due to the decrease in the magnitude of the first-order exchange rate of the slowly exchanged alpha-proton of D-alanine. Adding DL-alpha-glycerol 3-phosphate produced an increase in the stereospecificity of the second-order exchange rate observed with alanine but no significant change in the stereospecificity of the first-order exchange rate with tryptophan. The alpha-subunits are shown to increase the exchange rates of the alpha-protons of L-alanine and L-tryptophan. We conclude that the contribution of the R-group of an amino acid to the stereospecificity of the exchange reactions of its alpha-proton can be similar to or larger than that of its alpha-carboxylate group. Possible mechanisms that could explain the stereospecificity of these exchange reactions are discussed.

Factors affecting the stereospecificity and catalytic efficiency of the tryptophan synthase-catalysed exchange of the pro-2R and pro-2S protons of glycine.

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. The first- and second-order rate constants for exchange when the alpha 2 beta 2 enzyme complex is or is not saturated with glycine have been determined at pH 7.0 and 7.8. At pH 7.8 the effects of binding the allosteric effector, DL-alpha-glycerol 3-phosphate, and of removing the alpha-subunits have been examined. The beta-subunits preferentially catalyse the exchange of the pro-2R proton of glycine, but adding alpha-subunits decreases the stereospecificity of the exchange reactions. Likewise, binding of DL-alpha-glycerol 3-phosphate to the alpha 2 beta 2 enzyme complex causes a further decrease in the stereospecificity of this reaction. The stereospecificity of the second-order exchange reaction catalysed by the beta-subunits is 136-fold larger than that of the alpha 2 beta 2 enzyme complex in the presence of DL-alpha-glycerol 3-phosphate, while there is only a 5-fold decrease in the stereospecificity of the first-order exchange reaction under the same conditions. We discuss how these results relate to current theories which attempt to explain how the alpha-subunits and DL-alpha-glycerol 3-phosphate modify the catalytic properties of tryptophan synthase.

A comparative study of the kinetics and stereochemistry of the serine hydroxymethyltransferase- and tryptophan synthase-catalysed exchange of the pro-2R and pro-2S protons of glycine.

The stereospecificity of the serine hydroxymethyltransferase (EC 2.1.2.1)- and tryptophan synthase (EC 4.2.1.20)- catalysed exchange of the pro-2R and pro-2S alpha-protons of glycine was investigated by using 13C n.m.r. The exchange process is described in terms of a minimal four-step mechanism, and a method for analysing the exchange process by complete progress curves is presented. It is shown that serine hydroxymethyltransferase does not have absolute stereospecificity for the pro-2S-proton of glycine, but it catalyses the exchange of this proton 7400 times faster than the pro-2R proton of glycine. Tryptophan synthase is shown preferentially to catalyse the exchange of the pro-2R proton of glycine at a rate 380 times faster than the pro-2S proton of glycine. The exchange rates for the rapidly exchanged alpha-protons of glycine are similar for both enzymes. However, the exchange rates of the slowly exchanged alpha-protons differ by an order of magnitude. The structural features that may be responsible for the differences in the stereospecificity of the two enzymes are discussed.