title: A substrate-induced change in the stereospecificity of the serine-hydroxymethyltransferase-catalysed exchange of the alpha-protons of amino acids--evidence for a second catalytic site.
abstract: NMR has been used to study the catalysis of the hydrogen-deuterium exchange of the alpha-protons of amino acids by serine hydroxymethyltransferase (EC 2.1.2.1) from Escherichia coli. 13C-NMR was used to follow the exchange of the alpha-protons of [2-13C]glycine. The enzyme-catalysed first-order exchange rate of the pro-2S proton of glycine was approximately 7000 times more efficient than that of the pro-2R proton of glycine at both pH 7.0 and 7.8. 1H-NMR was used to follow the hydrogen-deuterium exchange rates of the alpha-protons of L- and D-2-amino derivatives of butyric, pentanoic and hexanoic acids at pH 7.8. Increasing the size of the R-group leads to a progressive change in the stereospecificity of the exchange reaction from the pro-2S proton of glycine to the 2R proton of L-amino acids. The stereospecificity for the alpha-protons of L-amino acids increased as the size of the R-group increased. With glycine, removal of tetrahydrofolate led to a large decrease in the stereospecificity of the exchange reaction but did not affect the exchange rates of the alpha-protons of any of the larger amino acids studied. We show that the Schiff base formed between L-2-aminohexanoic acid (L-norleucine) and pyridoxal 5'-phosphate binds at a different site from the Schiff base between glycine and pyridoxal 5'-phosphate. The molecular basis of these results is discussed.
authors.affiliation: Department of Biochemistry, University College Dublin, Ireland.
authors.forename: T B
authors.initials: TB
authors.lastname: Fitzpatrick
authors.forename: J P
authors.initials: JP
authors.lastname: Malthouse
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D001894:Borohydrides / Q000378:metabolism; D002087:Butyrates / Q000378:metabolism; D002208:Caproates / Q000378:metabolism / Q000494:pharmacology; D002247:Carbon Isotopes; D002384:Catalysis; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology*; D005998:Glycine / Q000378:metabolism / Q000494:pharmacology; D012696:Glycine Hydroxymethyltransferase / Q000378:metabolism*; D007700:Kinetics; D008968:Molecular Conformation; D010421:Pentanoic Acids / Q000378:metabolism; D011522:Protons; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012545:Schiff Bases / Q000378:metabolism; D013379:Substrate Specificity; D013763:Tetrahydrofolates / Q000378:metabolism
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D001894:Borohydrides; D002087:Butyrates; D002208:Caproates; D002247:Carbon Isotopes; D010421:Pentanoic Acids; D011522:Protons; D012545:Schiff Bases; D013763:Tetrahydrofolates; C030371:5,6,7,8-tetrahydrofolic acid; D011732:Pyridoxal Phosphate; D003903:Deuterium; C009282:sodium cyanoborohydride; D012696:Glycine Hydroxymethyltransferase; D005998:Glycine