α-thio-APS: A stereomechanistic probe of activated sulfate synthesis

Despite their broad application in the phosphoryl-transfer field, thio- nucleotides have not been available for the study of sulfate activation and transfer. There are two known forms of activated sulfate in the cell, APS (adenosine 5'-phosphosulfate) and PAPS (3'-phosphoadenosine 5'- phosphosulfate). PAPS is the only known sulfuryl group donor in metabolism, and sulfuryl transfer is used widely to regulate metabolism. This study presents the first synthesis of a thio-nucleotide analogue of activated sulfate, APS(α)S ((Sp)- and (Rp)-adenosine 5'-O-(1-thiophosphosulfate)). Two syntheses are described, one of which is a novel 'one-pot' method that is general for the site-specific delivery of the sulfuryl group. Both epimers of APS(α)S were purified and their stereochemical configurations were assigned. These compounds were used to address several stereomechanistic issues in the APS-synthesis reaction catalyzed by yeast ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4). The reaction is shown to proceed with inversion of configuration at the α-phosphorus (P(α)). The enzyme exhibits high selectivity for the (R(p)) epimer of APS(α)S when Mg²⁺ is the activating cation. The 'hard' vs 'soft' cation dependence of the enzyme's activity indicates that its selectivity is due to cation coordination at P(α). The absence of selectivity in the substrate binding reactions indicates that coordination at P(α) occurs after formation of the E·APS·PP(i)·M²⁺ complex.