Molecular Cloning, Expression and Site-Directed Mutagenesis of a Complementary DNA Encoding Rabbit Muscle Fructose 6-Phosphate-1-Kinase.

This dissertation contains three parts based on my research project which investigates three aspects of rabbit muscle phosphofructokinase (ATP: D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11; PFK). The first part reports the isolation and characterization of two full-length rabbit muscle PFK cDNAs. The DNA sequences of these two cDNAs (cDNA-A and cDNA-B) show an identical coding sequence but heterogeneous 5$\sp\prime$untranslated regions. cDNA-A is formed by removal of a 1.7 Kb upstream intron while cDNA-B retains the 3$\sp\prime$region of this intron. The second part describes the expression of the cDNA in several bacterial hosts. Recombinant rabbit muscle PFK has been expressed at a significant level in a PFK deficient E. coli strain DF1020 using the plasmid pPL2 as the expression vector. This expression system provides a visible selection for clones which express rabbit muscle PFK. The recombinant PFK resembles the enzyme purified from rabbit muscle with respect to its affinity for substrates, its allosteric behavior and its physical properties. The third part shows the structural and functional role of Gln-200 in rabbit muscle PFK (RMPFK). The comparison of the amino acid sequence of RMPFK to that of the PFK from Bacillus stearothermophilus suggests that Gln-200 of RMPFK is located in a region corresponding to the 6-F loop. This loop plays a critical role in the R-T transition of BsPFK. Mutation of RMPFK at position 200 affects the apparent affinity of the enzyme for Fru 6-P under the conditions for which RMPFK behaves allosterically. Three mutations were made at position 200: Gln-200 $\to$ Arg (Q200R), Gln-200 $\to$ Glu (Q200E), and Gln-200 $\to$ Ala (Q200A). The $\rm S\sb{0.5 (Fru 6-P)}$ of the Q200R mutant is 4-fold lower than that of the wild type RMPFK. The $\rm S\sb{0.5 (Fru 6-P)}$ of the Q200E mutant is increased about 14-fold. The Q200A mutant cannot achieve half-saturation, even at a concentration of Fru 6-P over 200 mM. Sigmoidal kinetics with respect to Fru 6-P were observed with Hill coefficients of 2.4, 1.9, and 1.0 for the wild type RMPFK and the Q200R and Q200E mutants, respectively. The data demonstrate that Gln-200 is a residue important for the cooperative binding of Fru 6-P to RMPFK