Impact on Catalysis of Secondary Structural Manipulation of the αC-Helix of Escherichia coli Dihydrofolate Reductase

Luyuan Li, Stephen J. Benkovic

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22 Scopus citations

Abstract

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

Original languageEnglish (US)
Pages (from-to)1470-1478
Number of pages9
JournalBiochemistry
Volume30
Issue number6
DOIs
StatePublished - Feb 1 1991

All Science Journal Classification (ASJC) codes

  • Biochemistry

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