The reactivity and function of cysteine residues in rabbit liver aldolase B.
Rabbit liver aldolase B (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) contains 8 SH groups/subunit and no disulfide bonds. In the native enzyme 3 SH groups/subunit are titrable with 5,5'-dithiobis(2-nitrobenzoic) acid (Nbs2), 2,2'-dithiodipyridine and N-ethylmaleimide, whereas p-mercuribenzoate is able to react with 4 thiol groups per subunit. Among the three thiol groups titrable with Nbs2, two react 'fast' with simple second-order kinetics, one reacts 'slow' and for this thiol group saturation kinetics is observed, suggesting a reversible binding of Nbs2 to the enzyme prior to covalent modification. It is shown that this binding most likely occurs via ionic interactions in the region close to the active site. The kinetic differentiation between the two 'fast' reacting groups is possible by kinetic analysis of the release of Nbs residues from the modified enzyme. Modification of all exposed SH groups of aldolase B results in 14-32% loss of enzymatic activity. The complete inactivation of liver aldolase by 1 mM p-mercuribenzoate reported previously (Waud, J.M., Feldman, E. and Schray, K.J. (1981) Arch. Biochem. Biophys. 206, 292-295) is shown to be caused by a nonspecific reaction of this reagent used in large excess. It is concluded that this isoenzyme differs from muscle aldolase in the reactivity of exposed SH groups, the mechanisms of the interaction with modifying agents and also in the effect of SH group modification on the enzymatic activity.
D000818:Animals; D003545:Cysteine; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000032:analysis; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D005634:Fructose-Bisphosphate Aldolase; D003545:Cysteine