The role of Lys272 in the pyridoxal 5-phosphate active site of Synechococcus glutamate-1-semialdehyde aminotransferase.
Glutamate-1-semialdehyde (GSA) aminotransferase catalyzes transfer of the C2 amino group of glutamate 1-semialdehyde to the C1 position to yield the tetrapyrrole precursor 5-aminolevulinate. Based on spectrophotometric and steady-state data, GSA aminotransferase is a B6-containing enzyme which uses a ping-pong bi-bi mechanism described for other aminotransferases. A putative active-site lysine at position 272 of Synechococcus GSA aminotransferase was replaced by Arg, Ile or Glu, and genes encoding the corresponding three site directed mutants were expressed in Escherichia coli. The catalytic competence of the resulting enzymes was determined. The similarity of the absorbance spectra of pyridoxal-P-treated forms of Lys272----Arg, Lys272----Ile, Lys272----Glu with free pyridoxal-P indicates that enzyme-bound pyridoxal-P does not form an internal aldimine in in these three site-directed mutants. Whereas Lys----Ile and Lys----Glu form only stable ketimines and aldimines with GSA and its analogues, addition of these compounds to the pyridoxamine-P and pyridoxal-P forms of Lys----Arg induces slow spectral changes, indicating the catalysis of a half-reaction with GSA, 4,5-dioxovalerate and 4,5-diaminovalerate. 5-Aminolevulinate apparently binds with both coenzyme forms of Lys272----Arg, however significant tautomeric rearrangement is only observed with the pyridoxal-P form. It is suggested that Lys272 is the covalent pyridoxal-P-binding site and that this catalytically active lysine residue channels the overall transamination reaction towards 5-aminolevulinate. The second-half reaction (4,5-diaminovalerate in equilibrium with 5-aminolevulinate) is possibly supported by the formation of an internal aldimine which correctly positions the C4 amino group of 4,5-diaminovalerate relative to the enzyme-bound pyridoxal-P.
Grimm
B
B
Department of Physiology, Carlsberg Laboratory, Copenhagen Valby, Denmark.
Smith
M A
MA
von Wettstein
D
D
D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D000458:Cyanobacteria / Q000201:enzymology*; D004269:DNA, Bacterial; D019751:Intramolecular Transferases; D007535:Isomerases / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011732:Pyridoxal Phosphate / Q000378:metabolism*
D004269:DNA, Bacterial; D011732:Pyridoxal Phosphate; D007535:Isomerases; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase; D008239:Lysine