Effects of point mutations at the flexible loop glycine-67 of Escherichia coli dihydrofolate reductase on its stability and function.


Abstract

To elucidate the role of a flexible loop (residues 64-72) in the stability and function of Escherichia coli dihydrofolate reductase, glycine-67 in this loop was substituted by site-directed mutagenesis with seven amino acids (Ala, Cys, Asp, Leu, Ser, Thr, and Val). The circular dichroism spectra suggested that the confirmation of the native structure was affected by the mutations in both the presence and absence of NADPH. The free energy change of unfolding by urea decreased in the order of G67A > G67S > or = wild-type > or = G67D > G67T > G67C > or = G67L > G67V. The steady-state kinetic parameters for the enzyme reaction, Km and kcat, were only slightly influenced, but the rate of the hydride transfer reaction was significantly changed by the mutations, as revealed by the deuterium isotope effect on the enzyme activity. These results suggest that site 67 in the flexible loop, being very far from the active site, plays an important role in the stability and function of this enzyme. The characteristics of the mutations were discussed in terms of the modified flexibility of the native structure, compared with the results of mutations at site 121 in another flexible loop. Study holds ProTherm entries: 2458, 2459, 2460, 2461, 2462, 2463, 2464, 2465 Extra Details: additive : EDTA(0.1 mM), point mutations; glycine; Escherichia coli dihydrofolate reductase;,stability; free energy; flexible loop

Submission Details

ID: TxWZ4wBV

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:19 p.m.

Version: 1

Publication Details
Ohmae E;Iriyama K;Ichihara S;Gekko K,J. Biochem. (1996) Effects of point mutations at the flexible loop glycine-67 of Escherichia coli dihydrofolate reductase on its stability and function. PMID:8743572
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Dihydrofolate reductase P0ABQ5 DYR_ECOL6
100.0 Dihydrofolate reductase P0ABQ4 DYR_ECOLI
100.0 Dihydrofolate reductase P0ABQ6 DYR_SHIFL
96.2 Dihydrofolate reductase P31073 DYR_CITFR
91.8 Dihydrofolate reductase P31074 DYR_KLEAE