Allosteric communication in mammalian muscle aldolase.
Mixed disulphide formation in the presence of oxidized glutathione reversibly inactivates rabbit skeletal muscle aldolase. Inactivation is allosteric, preferentially modifying Cys-72 on the surface of the aldolase homotetramer distant from active-site locations and subunit interfaces. Ion-exchange chromatography fractionates partly inactivated aldolase into three distinct enzymic species: unmodified enzyme, inactive fully modified enzyme corresponding to one thiol reacted per subunit, and inactive singly modified enzyme in which only one thiol has reacted. Acid-precipitable enzymic intermediates formed in the presence of substrate, D-fructose 1,6-bisphosphate, and product, dihydroxyacetone phosphate, indicates that active site binding is unaffected upon modification. The absence of enamine carbanion formation in the presence of substrate but not product is consistent with mixed disulphide formation's blocking -C-C- cleavage and/or subsequent D-glyceraldehyde 3-phosphate release. Inactivation upon single subunit modification and substrate protection against modification denotes that the blocked step is associated with a long-range conformational transition involving highly co-operative subunit behaviour.
Sygusch
J
J
Département de Biochimie/Médecine, Université de Montréal, CP 6128, Station Centre Ville, Montréal, QC, Canada H3C 3J7.
Beaudry
D
D
D000494:Allosteric Regulation / Q000502:physiology; D000818:Animals; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D003545:Cysteine / Q000737:chemistry; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005635:Fructosediphosphates / Q000378:metabolism; D019803:Glutathione Disulfide / Q000737:chemistry; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology*; D009154:Mutation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005635:Fructosediphosphates; D011994:Recombinant Proteins; D005986:Glyceraldehyde 3-Phosphate; D005634:Fructose-Bisphosphate Aldolase; D003545:Cysteine; C029063:fructose-1,6-diphosphate; D019803:Glutathione Disulfide