Impact on catalysis of secondary structural manipulation of the .alpha.C-helix of Escherichia coli dihydrofolate reductase

摘要: The alpha C-helix of Escherichia coli dihydrofolate reductase has been converted to its counterpart in Lactobacillus casei by a triple mutation the helix (H45R, W47Y, and I50F). These changes result 2-fold increase steady-state reaction rate (kcat = 26 s-1) that is limited an increased off for release tetrahydrofolate (koff 40 s-1 versus 12 s-1). On other hand mutant protein exhibits 10-fold KM value (6.8 microM) decrease hydride transfer (85 from NADPH dihydrofolate. elevated upon rebinding NADPH, characteristic wild-type enzyme-catalyzed reaction, diminished. intrinsic pKa (6.4) enzyme binary complex with similar wild type, but ternary 7.3, about on pH unit higher than value. Further mutagenesis (G51P insertion K52) was conducted incorporate hairpin turn unique C-terminus L. order adjust possible dislocation new helix. resultant pentamutant shows restoration many kinetic parameters, such as kcat (12 s-1), (1.1 microM dihydrofolate), khyd (526 values. synergism product also largely restored. A substrate-induced conformational change responsible fine tuning catalytic process found be associated newly installed structure. Asp27 residue reprotonated before release.