Refining the Half‐Site Reaction Mechanism of SULT1B1 Using Engineered Heterodimers

Abstract

The cytosolic sulfotransferases (SULTs) biotransform drugs and hormones into sulfate esters, aiding in the deactivation and/or excretion of the compounds from the cell and body. Each human SULT isoform is a homodimer interfacing along a conserved motif with the consensus sequence KxxxTVxxxE. Despite conservation of the dimerization domain across all fourteen human SULT isoforms, its contribution to SULT activity is not fully understood. Based on results from recent molecular dynamic simulations (MDS) of dimeric SULT1B1 in the presence and absence of the cofactor, 3′‐phosphoadenosine 5′phosphosulfate (PAPS), our laboratory has developed a mechanistic model for SULT dimer half‐site reactivity. In this proposed reaction mechanism, the binding of PAP(S) to the enzyme surface elicits structural changes which are communicated to the partnering subunit through the dimerization domain. The result of this communication is a dimer with a single active subunit at any given time. The dimer subunits oscillate between active and inactive states, “taking turns” as the active subunit. The advantage of this oscillating mechanism is thought to be an overall increased reaction rate, primarily propelled by a more favorable koff for the reaction byproduct, 3′ 5′‐diphosphoadenosine (PAP). As a means for testing the oscillating activity hypothesis in vitro, we sought to disrupt the activity of one subunit of the dimer while leaving the activity of the neighboring subunit fully in tact. Comparison of the heterodimer's kinetic parameters to those of the wildtype isoform would discern the involvement of the partnering subunit in the overall reaction mechanism. Two key amino acid residues were mutated to disrupt the activity of SULT1B1. Mutation of Arg258 to a Lys rendered the SULT deficient in its ability to bind PAPS (Kd ≈ 200 uM), whereas mutation of the catalytic base, His109, to a Tyr rendered the SULT incapable of catalyzing sulfonate transfer while leaving substrate binding unaffected. Unfortunately, the favorable kon for SULT dimerization complicated isolation of heterodimeric SULT species (only one subunit with the desired mutation). To overcome this limitation, we engineered SULT1B1 isoforms capable of selective dimerization, propelled by amino acid charge repulsion/attraction properties. This heterodimer‐generating system allowed us to manipulate each individual subunit independently prior to dimerization. Therefore, we combined the heterodimer‐generating system with the activity‐altering mutations (R258K and H109Y) to isolate SULT dimers with one fully active subunit. The resulting heterodimeric SULT isoforms are currently the subjects of in vitro sulfation activity analyses.Support or Funding InformationNIH Grants: GM113980 and ES022606