Aspartate aminotransferase with the pyridoxal-5'-phosphate-binding lysine residue replaced by histidine retains partial catalytic competence.
The active site residue lysine 258 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by means of site-directed mutagenesis. The mutant protein was expressed in Escherichia coli and purified to homogeneity. Addition of 2-oxoglutarate to its pyridoxamine form changed the coenzyme absorption spectrum (lambda max = 330 nm) to that of the pyridoxal form (lambda max = 330/392 nm). The rate of this half-reaction of transamination (kcat = 4.0 x 10(-4)s-1) is five orders of magnitude slower than that of the wild-type enzyme. However, the reverse half-reaction, initiated by addition of aspartate or glutamate to the pyridoxal form of the mutant enzyme, is only three orders of magnitude slower than that of the wild-type enzyme, kmax of the observable rate-limiting elementary step, i.e. the conversion of the external aldimine to the pyridoxamine form, being 7.0 x 10(-2)s-1. Aspartate aminotransferase (Lys258----His) thus represents a pyridoxal-5'-phosphate-dependent enzyme with significant catalytic competence without an active site lysine residue. Apparently, covalent binding of the coenzyme, i.e. the internal aldimine linkage, is not essential for the enzymic transamination reaction, and a histidine residue can to some extent substitute for lysine 258 which is assumed to act as proton donor/acceptor in the aldimine-ketimine tautomerization.
Ziak
M
M
Biochemisches Institut, Universität Zürich, Switzerland.
Jaussi
R
R
Gehring
H
H
Christen
P
P
D000818:Animals; D001219:Aspartate Aminotransferases / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D002645:Chickens; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006639:Histidine / Q000032:analysis*; D008239:Lysine / Q000032:analysis*; D009154:Mutation; D010957:Plasmids; D015202:Protein Engineering; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification
D011994:Recombinant Proteins; D006639:Histidine; D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases; D008239:Lysine