Adenosine-5'-phosphosulfate kinase from Penicillium chrysogenum: ligand binding properties and the mechanism of substrate inhibition.
[35S]Adenosine-5'-phosphosulfate (APS) binding to Penicillium chrysogenum APS kinase was measured by centrifugal ultrafiltration. APS did not bind to the free enzyme with a measurable affinity even at low ionic strength where substrate inhibition by APS is quite marked. However, APS bound with an apparent Kd of 0.54 microM in the presence of 5 mM MgADP. In the presence of 0.1 M (NH4)2SO4, Kd,app was increased to 2.1 +/- 0.7 microM. Bound [35S]APS was displaced by low concentrations of 3'-phosphoadenosine-5'-phosphosulfate (PAPS), or iso-(2') PAPS, or (less efficiently) by adenosine-3,5'-diphosphate (PAP) or adenosine-5'-monosulfate (AMS). The results support our conclusion that substrate inhibition of the fungal enzyme by APS results from the formation of a dead end E. MgADP.APS complex. That is, APS binds to the subsite vacated by PAPS in the compulsory (or predominately) ordered product release sequence (PAPS before MgADP). Radioligand displacement was used to verify the Kd for APS dissociation from E.MgADP.APS and to determine the Kd values for the dissociation of iso-PAPS (13 +/- 5 microM), PAP (4.8 mM), or AMS (5.2 mM) from their respective ternary enzyme.MgADP.ligand complexes. Incubation of the fungal enzyme with [gamma-32P]MgATP did not yield a phosphoenzyme that survives gel filtration or gel electrophoresis.
Renosto
F
F
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Martin
R L
RL
Segel
I H
IH
D000244:Adenosine Diphosphate / Q000378:metabolism; D000250:Adenosine Phosphosulfate / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D010408:Penicillium chrysogenum / Q000201:enzymology*; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D014461:Ultracentrifugation
D008024:Ligands; D010724:Phosphoadenosine Phosphosulfate; D000250:Adenosine Phosphosulfate; D000244:Adenosine Diphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase; D008274:Magnesium