Expression and functional analysis of the murine brain atp sulfurylase/adenosine 5'-phosphosulfate (APS) kinase enzyme a

The sulfate activation pathway in higher organisms consists of a bifunrtional ATP sulfurylàse/APS kinasc enzyme; in contrast, lower organisms have each activity on a separate polypeptide. ATP sulfurylase/ APS kinase isolated from rat chondrosarcoma has also been shown to use substrate channeling to efficiently transfer the labile APS intermediate to the kinase activity. We have cloned and expressed a cl)NA for niurine brain ATP sulfurylase/APS kinase in a bacterial expression system which yields a 66 kDa protein possessing both sulfurylase and kinase activity. The purified expressed protein has been shown to channel APS and demonstrates comparable kinetics to enzyme isolated from rat chondrosarcoma. Constructs encoding the two portions of the bifunctional enzyme, homologous to the separate enzymes found in lower organisms, were expressed to determine whether the domains could function independently. Although the APS kinase region did not yield a stable protein, the ATP sulfurylase fragment produced a stabie 44 kDa protein which exhibits sulfurylase activity. A second set of experiments used site-directed mutagenesis to elucidate residues involved in catalysis or stability. A series of mutations in the region encoding the ATP sulfurylase were expressed and His506Ala, Arg510Ala and Arg510Lys had no effect on either enzyme activity. In contrast, Cys77AIa, Arg421AIa, Arg421Lys, His425Ala, His 428Ala Arg522Ala, and Arg522Lys were found to greatly compromise ATP sulfury!ase activity while leaving kinasp activity intact and are therefore essential for enzyme function.

Duke Scholars

Author Andrea T. Deyrup Pathology