Sulfate-activating enzymes in normal and brachymorphic mice: evidence for a channeling defect.

Abstract

The severe reduction in the amount of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in cartilage from homozygous brachymorphic mice results from a decrease in the activities of both ATP sulfurylase (50%) and adenosine 5'-phosphosulfate (APS) kinase (14% of normal). In order to better understand the etiology of this double enzyme defect, a dual approach to elucidating the nature of the enzyme complex as well as its mechanistic properties was undertaken. Antibody reagents that react with both activities provide evidence for a single, bifunctional protein in both normal and mutant cartilage. Quantitative Western blot analysis indicates that a normal amount of a dysfunctional protein is produced in mutant cartilage. Kinetic studies show that the Vmax for mutant kinase is significantly reduced and that mutant sulfurylase and kinase appear to have lower KmAPS values than normal. Interestingly, the mutation appears to disrupt the channeling mechanism that has recently been demonstrated for this pathway [Lyle et al. (1994) Biochemistry 33, 6822-6827]. APS kinase from normal mouse cartilage utilizes APS supplied by ATP sulfurylase much more efficiently than APS which is added exogenously; i.e., channeling efficiency is > 90%. In contrast, the mutant enzymes exhibit only 54% channeling efficiency. Lastly, isotope dilution and enrichment experiments show directly that the APS binding sites of the mutant enzymes are more accessible to free APS than are those of the normal enzymes. These data suggest that the mutation primarily affects the catalytic properties of the PAPS activation system by altering the function of the novel coupling mechanism between the two activities, causing a decrease in the ability to channel APS and produce PAPS efficiently.