Enzymic Hydrolysis of 3‐Acetyl‐estrogens or Analogues by Glutamate Dehydrogenase with Specific Acylation of the Estrogen Binding Site

The estrogen analogues or derivatives 4‐acetyldiethylstilbestrol, 3‐acetyl‐estradiol and 3‐acetyl‐2‐nitroestrone were hydrolysed by glutamate dehydrogenase with covalent incorporation of an acetyl group into the enzyme molecule, probably through a transacylation process. When 3‐acetyl‐2‐nitroestrone was used as a ‘substrate’, spectrophotometric titration of the free product (a nitrophenol) allowed kinetic studies: there was a linear relationship between initial hydrolysis rates and enzyme concentration and Michaelis kinetics were observed suggesting an enzyme catalyzed process. The covalent incorporation of the acetyl group on the enzyme was studied using 3‐[2′‐14C]acetyl‐2‐nitroestrone: in the absence of NADH, lysine residues 419 and 422 were specifically acetylated without enzyme inactivation. In the presence of NADH, the labelling was distributed over two more lysine residues, 143 and 155, with progressive inactivation. Estradiol lowered the hydrolysis rate of 3‐acetyl‐2‐nitroestrone by glutamate dehydrogenase, protected the enzyme against acylation and against the inactivation which is observed in the presence of NADH. Moreover glutamate dehydrogenase acylated by 3‐acetyl‐2‐nitroestrone became less sensitive to its allosteric effector: estradiol. Thus, it is suggested that hydrolysis of acetyl‐estrogen or analogues by glutamate dehydrogenase and acylation of the enzyme occur in or near the estrogen binding site.