Induction of positive cooperativity by amino acid replacements within the C-terminal domain of Penicillium chrysogenum ATP sulfurylase

Abstract

ATP sulfurylase from Penicillium chrysogenum is an allosteric enzyme in which Cys-509 is critical for maintaining the R state. Cys-509 is located in a C-terminal domain that is 42% identical to the conserved core of adenosine 5'-phosphosulfate (adenylylsulfate) (APS) kinase. This domain is believed to provide the binding site for the allosteric effector, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Replacement of Cys-509 with either Tyr or Ser destabilizes the R state, resulting in an enzyme that is intrinsically cooperative at pH 8 in the absence of PAPS. The kinetics of C509Y resemble those of the wild type enzyme in which Cys-509 has been covalently modified. The kinetics of C509S resemble those of the wild type enzyme in the presence of PAPS. It is likely that the negative charge on the Cys-509 side chain helps to stabilize the R state. Treatment of the enzyme with a low level of trypsin results in cleavage at Lys-527, a residue that lies in a region analogous to a PAPS motif-containing mobile loop of true APS kinase. Both mutant enzymes were cleaved more rapidly than the wild type enzyme, suggesting that movement of the mobile loop occurs during the R to T transition.