Date of Award

9-18-1998

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

John B. Shabb

Abstract

The carboxyl terminal 19 amino acids of the type $\rm I\alpha$ regulatory subunit of cAMP-dependent protein kinase were investigated to determine their contribution to cAMP selectivity. The parent $\rm RI\alpha$ subunit contained an Ala-to-Thr mutation at position 334 so that it would bind both cAMP and cGMP with high-affinity. Stop codons were introduced into the parent cDNA construct at positions corresponding to Val-375, Asn-372, Gln-370, and Cys-360. The proteins were expressed in E. coli and purified by affinity chromatography, as were all other proteins in this study. Truncation mutants were initially characterized for cAMP and cGMP dissociation properties. This was done to determine which residues in the carboxyl terminus were minimally required for high-affinity binding and/or selective binding of cAMP. Site-selective cAMP analogs were used to compete against $\rm\lbrack \sp3H\rbrack $ cAMP binding to the mutant $\rm RI\alpha$ subunits to correctly assign fast and slow dissociation $\rm t\sb{1/2}$ values to the A and B domains. A greater than sixty-fold drop in B domain $\rm t\sb{1/2}$ values in the Asn-372-stop to Gln-370-stop transition implicated Tyr-371 as an important cAMP-binding determinant. A similar drop in $\rm \lbrack \sp3H\rbrack $ cGMP $\rm t\sb{1/2}$ values for the same transition suggested that the cGMP/cAMP selectivity was not altered. To test this further, Tyr-371 in the parent construct was mutated to Ala, Phe, and Arg. The cAMP and cGMP $\rm t\sb{1/2}$ values were determined, as were protein kinase activation constants $\rm (K\sb{a})$ for holoenzymes formed from mutant $\rm RI\alpha$ subunits and purified catalytic subunit. The $\rm K\sb{a}$ data suggested that mutation of Tyr-371 enhanced B domain selectivity. Isolated B domains containing Tyr-371-Arg or Tyr-371-Phe mutations were constructed, expressed and purified to determine their relative inhibition constants $\rm (K\sp\prime\sb{I})$ for cGMP vs. cAMP. These data showed that B domain cAMP selectivity was minimally affected by alteration of Tyr-371. Based on these results, it is concluded that aromatic stacking is not important for determining B domain cyclic nucleotide selectivity. It is proposed that the main function of Tyr-371 is stabilization of the B domain cAMP-binding pocket through hydrogen bonding with Glu-324.

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