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© 1994 Oxford University Press

OTHER

Sequence, expression and characterization of HPRTMoose Jaw: a point mutation resulting in cooperativity and decreased substrate affinities

Therese Lightfoot1,2, Raymond M. Lewkonla3 and Floyd F. Snyder1,2,*

1Departments of 1Paediatrics 2Medical Biochemistry 3Medidne, Faculty of Medicine, 3330 Hospital Drive N W., University of Calgary Calgary, Alberta TN2 4N1, Canada

* To whom correspondence should be addressed

Received April 21, 1994; Accepted June 13, 1994

We have sequenced and studied the expressed protein of an HPRT mutation characterized by 5–12% residual erythrocyte activity, for which affected males exhibit hyperuricemia, arthritis and renal disease but are without severe neurological Involvement. The HPRTMoose Jaw mutation is due to a single C to G transverslon at nucleotlde 582 relative to Initiation of translation corresponding to substitution of aspartate 194 by glutamate. The mutant and wild type proteins were expressed and purified using the bacterial expression vector, pMAL-c2. The Km for hypoxanthlne was Increased 12-fold from 0.94 ± 0.26 to 11.5 ± 1.3 µM for control and mutant respectively. The apparent Km for PP-rlbose-P was Increased 44-fold from 6.8 ± 0.6 to 295 ± 7 µM for control and mutant respectively. Although the Kcat of the mutant protein was equivalent to wild type, the catalytic efficiency, Kcat/Km, of the purified mutant protein was only 6 and 3% of wild type with hypoxanthine and PP-rlbose-P respectively. The mutant protein also exhibited positive cooperativity with PP-ribose-P, having a Hill coefficient of 2.3. The decreased substrate affinities and PP-ribose-P associated cooperativity of HPRTMoose Jaw provide additional evidence for the influence of carboxy-terminal residues of HPRT in specific catalytic functions.


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