Recent studies on the tissue distribution and developmental regulation of ADA activity in mice show that very high ADA levels exist in the murine alimentary tract (tongue, esophagus, forestomach, proximal small intestine) and at the fetal-maternal interface. Levels of three other enzymes involved in purine catabolism--purine nucleoside phosphorylase (PNP), guanine deaminase (GDA), and xanthine dehydrogenase (XDH)--were measured and compared with ADA activity. The highest levels of PNP, GDA and XDH were found in the proximal small intestine. Levels of these three enzymes were much lower in the tongue, esophagus, forestomach and fetal-maternal interface in marked contrast to ADA distribution. Tissue-specific differences in PNP, XDH and ADA activity correlated with RNA abundance indicating that the regulation of gene expression is at the level of mRNA production. Thus, ADA is part of a purine catabolic pathway leading to the production of uric acid that is present at the highest known level in the proximal small intestine. ADA may have additional roles in other tissues.
We also studied catalytic aspects of ADA. The proposed catalytic mechanism of ADA based on the recent elucidation of its X-ray structure (Wilson et al., Science 252:1278) hypothesized that Glu$\sp{217}$ was involved in protonation of N-1 of the adenosine ring, suggesting a vital role for this residue in tetrahedral intermediate formation. To study the importance of this residue, Glu$\sp{217}$ of murine ADA was mutated to Asp, Gly, Gln and Ser. A novel purification protocol without the use of affinity chromatography was developed. Circular dichroism and zinc analysis showed no change in secondary structure or zinc content, respectively, compared to the native protein. The mutants showed only slight variation in K$\sb{\rm m}$ but dramatically reduced k$\sb{\rm cat}$ ranging from 0.03% (E217G) to 1% (E217S) of wild type activity. K$\sb{\rm i}$'s with purine riboside were similar for wild-type and mutant adenosine deaminases further underscoring the minimal role this residue plays in substrate binding. A characteristic UV difference spectrum of native ADA with purine riboside, indicative of protonation of the purine ring at N-1 of the substrate, was not observed with the mutants. These data confirm the importance of Glu$\sp{217}$ in catalysis as suggested by the crystal structure of mADA.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/16758 |
| Date | January 1994 |
| Creators | Mohamedali, Khalid Amanali |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | application/pdf |
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