CHEMICAL MODIFICATION OF BILE ACID-COA LIGASE

The effect of chemical modification of bile acid: CoA ligase on its en zymatic activity was examined. Reagents which modify tyrosine and carb oxyl groups did not affect the activity of either the purified enzyme or the enzyme in its native microsomal environment. The modification o f arginine residues with either diacetyl or phenylglyoxal resulted in a loss of activity for both the purified and microsomal forms of the e nzyme. ATP was able to protect the enzyme from inactivation. Neither c holate nor CoA were able to alter the time-course of inactivation. The sulfhydryl reagent N-ethylmaleimide (MalNEt) produced a biphasic effe ct on both the purified and microsomal forms of the enzyme. At short r eaction times ligase activity increased, but further reaction lead to nearly complete inactivation. With the purified enzyme, ATP increased the extent of activation by MalNEt and decreased the rate of inactivat ion. With microsomes, ATP did not affect the extent of activation by M alNEt, but did slow the rate of inactivation. For both the purified an d microsomal forms cholate provided no protection. Treatment of both f orms of the enzyme with the sulfhydryl reagent iodoacetic acid produce d a similar biphasic activation/inactivation of the ligase. It was hyp othesized that modification of a fast-reacting cysteine leads to activ ation while a slower-reacting cysteine leads to inactivation. This lat ter cysteine appeared to be in the ATP-binding site on the enzyme.