Site-specific methylation of a strategic lysyl residue in aspartate aminotransferase.
Conditions for reductive methylation of amine groups in proteins using formaldehyde and cyanoborohydride can be chosen to modify selectively the active site lysyl residue of aspartate aminotransferase among the 19 lysyl residues in each subunit of this protein. Apoenzyme must be treated, under mildly acidic conditions (pH = 6), at a relatively low molar ratio of formaldehyde to protein (40:1); and, upon reduction with sodium cyanoborohydride, 85% of the formaldehyde is incorporated at Lysine 258 and 15% at the amino-terminal alanyl residue. The modified protein, characterized after tryptic hydrolysis, separation of the peptides by high performance liquid chromatography procedures and subsequent amino acid analysis, shows that lysine 258 is preferentially modified as a dimethylated derivative. Modified apoenzyme can accept and tightly bind added coenzyme pyridoxal phosphate, as measured by circular dichroism procedures. The methylated enzyme is essentially catalytically inactive when measured by standard enzymatic assays. On the other hand, addition of the substrate, glutamate, produces the characteristic absorption spectral shifts for conversion of the active site-bound pyridoxal form of the coenzyme (absorbance at 400 nm) to its pyridoxamine form (absorbance at 330 nm). Such a half-transamination-like process occurs as in native enzyme, albeit at several orders of magnitude lower rate. This event takes place even though the characteristic internal holoenzyme Schiff's base between Lys-258 and aldehyde of bound pyridoxal phosphate does not exist in methylated, reconstituted holoenzyme. It is concluded that this chemically transformed enzyme can undergo a half-transamination reaction with conversion of active site-bound coenzyme from a pyridoxal to a pyridoxamine form, even when overall catalytic turnover transamination cannot be detected.
D000595:Amino Acid Sequence; D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D002942:Circular Dichroism; D007527:Isoenzymes / Q000378:metabolism; D008239:Lysine; D008745:Methylation; D009206:Myocardium / Q000201:enzymology*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011487:Protein Conformation; D013552:Swine; D014357:Trypsin
D007527:Isoenzymes; D010446:Peptide Fragments; D001219:Aspartate Aminotransferases; D014357:Trypsin; D008239:Lysine