INVOLVEMENT OF PHE(19) IN THE MN2-L-MALATE BINDING AND THE SUBUNIT INTERACTIONS OF PIGEON LIVER MALIC ENZYME()

A triple mutant, F19S/N250S/L353Q, of pigeon liver malic enzyme was fo und to have no detectable enzymatic activity [Chou, W.-Y., Huang, S.-M ., & Chang, G.-G. (1994) Arch. Biochem. Biophys. 310, 158-166]. In the present study, point mutants at these positions (F19S, N250S, and L35 3Q) were prepared by site-directed mutagenesis. Both N250S and L353Q h ave kinetic properties similar to those of the wild-type. On the other hand, the K-m(app) values for both Mn2+ and L-malate of F19S were inc reased by approximately 10-fold, while the k(cat) value was decreased by 5-fold, which results in a decrease of the apparent catalytic effic iency (k(cat)/KmNADPKmMalKmMn) by approximately 300-fold. These result s clearly indicate that the F19S mutation is mainly responsible for th e undetectable enzyme activity of the triple mutant. Three more Phe(19 ) mutants (F19Y, F19G, and F19A) were then prepared. There is a direct correlation between the size of the substitutes and the affinities fo r Mn2+ and L-malate. The kinetic parameters for F19Y were similar to t hose for wild-type. Both F19A and F19G reveal a 5-fold decrease of k(c at) values. Two K-dMn values for the high- and low-affinity sites, res pectively, were detectable for the wild-type. On the contrary, only on e K-dMn value was detected for the F19 mutants, which was increased in the order of F19G > F19A > F19S > F19Y, with F19G being the most affe cted mutant. The K-mMal values of F19G and F19A were increased 100- an d 6-fold, respectively. The catalytic efficiency (k(cat)/KmNADPKdMalKd Mn) of F19G was decreased to only 0.01% of that of the wild-type. The above results clearly indicate that the hydrophobic aromatic ring at p osition 19 plays a critical role in L-malate and Mn2+ binding. Further more, all mutants that have a small residue at position 19 exist as mo nomers. Therefore, Phe(19) may locate in or near the regions for Mn2+- L-malate binding as well as for the subunit contact. These results are compatible with the asymmetric model for the quaternary structure of malic enzyme we proposed previously [Chang, G.-G., Huang, T.-M., Huang , S.-M., & Chou, W.-Y. (1994) Eur. J. Biochem. 225, 1021-1027]. The po ssible roles of the N-terminus of malic enzyme were also addressed.