PMID: 8454578Jan 1, 1993Paper

Effects of point mutation in a flexible loop on the stability and enzymatic function of Escherichia coli dihydrofolate reductase

Journal of Biochemistry
K GekkoM Iwakura

Abstract

To elucidate the role of a flexible loop in the stability and function of Escherichia coli dihydrofolate reductase, glycine-121 in the flexible loop (117-131) was substituted to valine and leucine by site-directed mutagenesis. Despite the increased hydrophobicity of the side chains, the free energy changes of unfolding of the two mutants (G121V and G121L) determined by urea denaturation at 15 degrees C were decreased by 1.22 and 0.38 kcal/mol, respectively, compared with that of the wild-type. Thermal denaturation temperature, as monitored by differential scanning calorimetry, was decreased by 2.4 and 5.2 degrees C for G121V and G121L, respectively, accompanying the decrease in enthalpy change of denaturation. These findings indicate that the structure of DHFR is destabilized by the mutations, predominantly due to the large decrease in enthalpy change of denaturation relative to entropy change of denaturation. The steady-state kinetic parameter in the enzyme reaction, Km, was not influenced but kcat was greatly decreased by these mutations, resulting in 240- and 52-fold decreases in kcat/Km for G121V and G121L, respectively. The main effect of the mutations appeared to be modification of the flexibility of the loop due to overc...Continue Reading

Citations

Apr 1, 1996·Journal of Biomolecular Structure & Dynamics·Z FuF Takusagawa
Jul 1, 1997·Protein Science : a Publication of the Protein Society·H HuangR T Borchardt
Jan 1, 1997·Protein Science : a Publication of the Protein Society·J S FetrowR E Burton
Oct 7, 1998·Protein Science : a Publication of the Protein Society·A J ConstansJ T Lecomte
Apr 10, 2015·Physical Chemistry Chemical Physics : PCCP·Louis Y P LukRudolf K Allemann
Aug 28, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Morgan B VaughnR Brian Dyer
Jul 12, 2014·Biochemistry·Enas M BehiryRudolf K Allemann
Jun 14, 2013·Biochemistry·David D BoehrPeter E Wright
Dec 30, 2014·Accounts of Chemical Research·Gira BhabhaJames S Fraser
Mar 21, 2017·Biochemistry·E Joel LoveridgeRudolf K Allemann
Feb 24, 2016·Biochemistry·Michael J ReddishR Brian Dyer
Feb 7, 2019·Biochemistry·Michael R DuffElizabeth E Howell
Jul 25, 2018·The Journal of Physical Chemistry. B·Anil R MhashalDan Thomas Major

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