Structure−Function Modeling of the Interactions of N-Alkyl-N-hydroxyanilines with Rat Hepatic Aryl Sulfotransferase IV

Abstract

Although previous investigations have clearly shown that N-hydroxy arylamines and N-hydroxy heterocyclic amines are substrates for sulfotransferases, relatively little is known about which structural features of the N-hydroxy arylamines are important for sulfation to occur. The purpose of this investigation was to determine the extent to which secondary N-alkyl-N-hydroxy arylamines interact with aryl sulfotransferase (AST) IV (also known as tyrosine-ester sulfotransferase or ST1A1) and to evaluate these interactions using molecular modeling techniques. AST IV is a major cytosolic sulfotransferase in the rat, and it catalyzes the sulfation of various phenols, benzylic alcohols, arylhydroxamic acids, oximes, and primary N-hydroxy arylamines. In this study, three secondary N-hydroxy arylamines, N-hydroxy-N-methylaniline, N-ethyl-N-hydroxyaniline, and N-hydroxy-N-n-propylaniline, were found to be substrates for the purified rat hepatic AST IV. However, when the N-alkyl substituent was an n-butyl group (i.e., N-n-butyl-N-hydroxyaniline), the interaction with the enzyme changed from that of a substrate to competitive inhibition. This change in specificity was further explored through the construction and use of a model for AST IV based on mouse estrogen sulfotransferase, an enzyme whose crystal structure has been previously determined to high resolution. Molecular modeling techniques were used to dock each of the above N-hydroxy arylamines into the active site of the homology model of AST IV and determine optimum ligand geometries. The results of these experiments indicated that steric constraints on the orientation of binding of secondary N-alkyl-N-hydroxy arylamines at the active site of AST IV play a significant role in determining the nature of the interaction of the enzyme with these compounds.

Comparative Molecular Field Analysis of Substrates for an Aryl Sulfotransferase Based on Catalytic Mechanism and Protein Homology Modeling

“…As a result of this progress made in the determination of sulfotransferase structures and the common structural features of all sulfotransferase structures examined to date, we have conducted homology modeling and substrate/inhibitor-docking studies on AST IV. 19 These crystal structures have also contributed to our knowledge of the sulfotransferase reaction mechanism. 38 Cocrystallization of mEST with the PAP-vanadate complex as a transition state mimic, coupled with mutational analysis, confirmed that the sulfotransferase-catalyzed reaction proceeds by an in-line sulfuryl transfer reaction from PAPS to the substrate.…”

“…Many catecholamines, 11 tyrosine esters, 11 peptides with N-terminal tyrosines, 11 hydroxamic acids, 11,[18][19][20][21] oximes, 22,23 benzylic alcohols, 17,24 and nitroalkanes 14,15,25 are substrates for the enzyme. Sulfuric acid esters of several AST IV substrates, such as those derived from some arylhydroxamic acids, benzylic alcohols, and N-hydroxyarylamines, are known to give rise to cellular necrosis and chemical carcinogenesis.…”

Influence of Phenylalanines 77 and 138 on the Stereospecificity of Aryl Sulfotransferase Iv

“…Computational studies on 1,2,3,4-tetrahydro-1-naphthol and 2-naphthol in the active site of the rat AST IV were performed using the SYBYL molecular modeling package (SYBYL version 6.7; Tripos Inc., St. Louis, MO) running on a Silicon Graphics O2 workstation (SGI, Mountain View, CA). A previously developed homology model of AST IV (King et al, 2000) was used as the basis for all docking and energy minimization calculations. Coordinates for modeling an estradiol molecule at the sulfuryl acceptor site were obtained from the structure of a mouse estrogen sulfotransferase-17␤-estradiol-PAP complex (PDB crystal structure code: 1AQU) (Kakuta et al, 1997).…”

“…A homology model for AST IV has been previously developed (King et al, 2000) based on the electron density map of mouse estrogen sulfotransferase (Kakuta et al, 1997). Mouse estrogen sulfotransferase serves as a particularly good structure for homology modeling of AST IV due to the availability of crystal structures that include enzyme-substrate and enzyme-product complexes as well as the structure of an enzyme-PAPvanadate complex that serves as a transition state model (Kakuta et al, 1998).…”

“…The starting point for these studies involved the use of a previously developed homology model for the three-dimensional structure of this phenol sulfotransferase (King et al, 2000), and preliminary molecular docking experiments led to the hypothesis that one or more of three aromatic residues, Phe77, Phe138, or Tyr236, might be important in determining steric interactions with chiral benzylic alcohols leading to stereospecificity. Although the part of the original hypothesis relating to Tyr236 was disproved, the retention of stereospecificity upon alteration of this aromatic residue that is near the docked substrate in the homology model is significant, since it serves as a control experiment that highlights the specificity of the interactions of the other two aromatic residues in this region of the sulfotransferase.…”