IDENTIFICATION OF KEY AMINO ACIDS RESPONSIBLE FOR THE SUBSTANTIALLY HIGHER AFFINITIES OF HUMAN TYPE 1 3β-HYDROXYSTEROID DEHYDROGENASE/ISOMERASE (3β-HSD1) FOR SUBSTRATES COENZYMES AND INHIBITORS RELATIVE TO HUMAN 3β-HSD2

摘要

The human type 1 (placenta, breast tumors, prostate tumors) and type 2 (adrenals, gonads) isoforms of 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD1 and 3β-HSD2) are encoded by two distinct genes that are expressed in a tissue-specific pattern. Our recent studies have shown that His156 contributes to the 14-fold higher affinity that 3β-HSD1 exhibits for substrate and inhibitor steroids compared to human 3β-HSD2 containing Tyr156 in the otherwise identical catalytic domain. Our structural model of human 3β-HSD localizes His156 or Tyr156 in the subunit interface of the enzyme homodimer. The model predicts that Gln105 on one enzyme subunit has a higher probability of interacting with His156 on the other subunit in 3β-HSD1 than with Tyr156 in 3β-HSD2. The Q105M mutant of 3β-HSD1 (Q105M1) shifts the Michaelis-Menten constant (Km) for 3β-HSD substrate and inhibition constants (Ki) for epostane and trilostane to the much lower affinity profiles measured for wild-type 3β-HSD2 and H156Y1. However, the Q105M2 mutant retains substrate and inhibitor kinetic profiles similar to those of 3β-HSD2. Our model also predicts that Gln240 in 3β-HSD1 and Arg240 in 3β-HSD2 may be responsible for the 3-fold higher affinity of the type 1 isomerase activity for substrate steroid and cofactors. The mutation increases the isomerase substrate Km by 2.2-fold to a value similar to that of 3β-HSD2 isomerase and abolishes the allosteric activation of isomerase by NADH. The R240Q2 mutation converts the isomerase substrate, cofactor and inhibitor kinetic profiles to the 4- to 14-fold higher affinity profiles of 3β-HSD1. Thus, key structural reasons for the substantially higher affinities of 3β-HSD1 for substrates, coenzymes and inhibitors have been identified. These structure and function relationships can be used in future docking studies to design better inhibitors of the 3β-HSD1 that may be useful in the treatment hormone-sensitive cancers and preterm labor.