Chemical trapping of complexes of dihydroxyacetone phosphate with muscle fructose-1,6-bisphosphate aldolase.
Dihydroxyacetone phosphate (DHAP) in equilibrium with FDP aldolase of muscle is present in the form of two major covalent complexes. One, representing approximately 60% of total bound substrate, decomposes to Pi and methylglyoxal upon acid denaturation of the enzyme as first reported by Grazi and Trombetta [Grazi, E., & Trombetta, G. (1979) Biochem. J. 175, 361-365]. This is now shown to be the enzyme-eneamine phosphate reaction intermediate since Pi formation is prevented if the acid denaturation is done in the presence of potassium ferricyanide, an oxidant of the eneamine. The enzyme-eneamine aldehyde X Pi 6, presumed to be an intermediate of the slow methylglyoxal synthetase reaction of aldolase, must not be a significant source of the Pi produced upon denaturation and is probably not a significant component of the equilibrium. The oxidation product, the enzyme-imine of phosphopyruvaldehyde, is sufficiently stable in 1 N HCl, t1/2 = 76 min at 0 degree C, to be isolated with the trichloroacetic acid precipitated protein. A second covalent complex, approximately 20-24% of bound dihydroxyacetone [32P]phosphate, remains with the protein during acid denaturation and centrifugation. This acid-stable complex is formed rapidly and is chased rapidly by unlabeled substrate. Its stability in 1 N HCl is similar to that of the ferricyanide-oxidized derivative mentioned above. From this and its reactivity with cyanoborohydride in acid, this complex is thought to be the imine adduct of DHAP with aldolase 4 and/or the carbinolamine complex 3 present in the initial equilibrium. D-Glyceraldehyde 3-phosphate in the carbonyl form also forms an acid-precipitable complex with aldolase.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D004099:Dihydroxyacetone Phosphate / Q000378:metabolism*; D005292:Ferricyanides / Q000494:pharmacology; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011489:Protein Denaturation; D011817:Rabbits; D014306:Trioses / Q000378:metabolism*
D005292:Ferricyanides; D014306:Trioses; C007931:hexacyanoferrate III; D005986:Glyceraldehyde 3-Phosphate; D004099:Dihydroxyacetone Phosphate; D005634:Fructose-Bisphosphate Aldolase