Paracatalytic self-inactivation of fructose-1,6-bisphosphate aldolase. Structure of the crosslink formed at the active site.
Oxidation of enzyme-substrate carbanion intermediates by extrinsic oxidants may result in irreversible paracatalytic inactivation of certain enzymes. In paracatalytically modified fructose-1,6-bisphosphate aldolase from rabbit muscle the polypeptide chain had been found to be crosslinked at active-site Lys229 (Schiff base forming with substrate) and Lys146 by a phosphorylated three-carbon moiety [Lubini, D. G. E. and Christen, P. (1979) Proc. Natl Acad. Sci. USA 76, 2527-2531]. In the present study, the structure of this crosslink was elucidated by instrumental analysis. Aldolase was paracatalytically modified in the presence of fructose 1,6-bisphosphate and hexacyanoferrate(III). The completely inactivated enzyme was digested with pronase. The crosslinked peptide was isolated by gel filtration and reverse-phase HPLC. Mass spectroscopy, 1H- and 13C-NMR showed that a derivative of dihydroxyacetone phosphate forms an amidine with the epsilon-amino groups of the two lysine residues: [formula: see text]
Gupta
S
S
Biochemisches Institut der Universität Zürich, Switzerland.
Hollenstein
R
R
Kochhar
S
S
Christen
P
P
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003432:Cross-Linking Reagents; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D015394:Molecular Structure; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry; D011402:Pronase / Q000378:metabolism; D011817:Rabbits; D017421:Sequence Analysis
D000596:Amino Acids; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D011402:Pronase; D005634:Fructose-Bisphosphate Aldolase; D008239:Lysine