A SUBSTRATE-INDUCED CHANGE IN THE STEREOSPECIFICITY OF THE SERINE-HYDROXYMETHYLTRANSFERASE-CATALYZE EXCHANGE OF THE ALPHA-PROTONS OF AMINO-ACIDS - EVIDENCE FOR A 2ND CATALYTIC SITE

NMR has been used to study the catalysis of the hydrogen-deuterium exc hange of the alpha-protons of amino acids by serine hydroxymethyltrans ferase (EC 2.1.2.1) from Escherichia coli. C-13-NMR was used to follow the exchange of the a-protons of [2-C-13]glycine. The enzyme-catalyse d first-order exchange rate of the pro-2S proton of glycine was approx imate to 7000 times more efficient than that of the pro-2R proton of g lycine at both pH 7.0 and 7.8. H-1-NMR was used to follow the hydrogen -deuterium exchange rates of the alpha-protons of L- and D-2-amino der ivatives of butyric, pentanoic and hexanoic acids at pH 7.8. Increasin g the size of the R-group leads to a progressive change in the stereos pecificity of the exchange reaction from the pro-2S proton of glycine to the 2R proton of L-amino acids. The stereospecificity for the a pro tons of L-amino acids increased as the size of the R-group increased. With glycine, removal of tetrahydrofolate led to a large decrease in t he stereospecificity of the exchange reaction but did not affect the e xchange rates of the alpha-protons of any of the larger amino acids st udied. We show that the Schiff base formed between L-2-aminohexanoic a cid (L-norleucine) and pyridoxal 5'-phosphate binds at a different sit e from the Schiff base between glycine and pyridoxal 5'-phosphate. The molecular basis of these results is discussed.