1993
DOI: 10.1021/bi00058a002
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Titration of histidine 62 in R67 dihydrofolate reductase is linked to a tetramer .tautm. two-dimer equilibrium

Abstract: R67 dihydrofolate reductase (DHFR) is an R-plasmid encoded protein that confers clinical resistance to the antibacterial drug trimethoprim. To determine whether an acidic titration in kinetic pH profiles is related to titration of histidines 62, 162, 262, and 362, the stability of tetrameric R67 DHFR has been monitored as a function of pH. For the pH range 5-8, tetrameric R67 DHFR reversibly dissociates into dimers, as monitored by ultracentrifugation and molecular sieving techniques. From the crystal structur… Show more

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Cited by 38 publications
(101 citation statements)
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“…This suggests that the latter must not be simply an addition of the two mechanisms such as U i extended M i closed M, where U is unfolded monomer; extended M is a "folded" monomer where only the AB and CD interfaces interact; and closed M is the monomeric species of quadruple R67 DHFR that corresponds to the active, native tetrameric species. The folded state at pH 5 must not approximate a folding intermediate at pH 8 (12). Thus, even if a heterotetramer were constructed, it would quickly reequilibrate into a mixture of hetero-and homospecies at pH values used to monitor catalysis.…”
Section: Discussionmentioning
confidence: 99%
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“…This suggests that the latter must not be simply an addition of the two mechanisms such as U i extended M i closed M, where U is unfolded monomer; extended M is a "folded" monomer where only the AB and CD interfaces interact; and closed M is the monomeric species of quadruple R67 DHFR that corresponds to the active, native tetrameric species. The folded state at pH 5 must not approximate a folding intermediate at pH 8 (12). Thus, even if a heterotetramer were constructed, it would quickly reequilibrate into a mixture of hetero-and homospecies at pH values used to monitor catalysis.…”
Section: Discussionmentioning
confidence: 99%
“…The molar extinction coefficient used to assess enzymic reduction of DHF was 12,300 M Ϫ1 cm Ϫ1 (11). pH Dependence of Oligomerization State-To monitor a pH-dependent equilibrium between tetramer and two dimers in native R67 DHFR or an equivalent dimer i 2 monomers transition in double R67 DHFR or an equivalent monomer i altered monomer conformational change in quadruple R67 DHFR, tryptophan fluorescence was monitored as a function of pH (6,12). The emission spectra for the DHFRs (excitation, 295 nm) were measured from 300 to 450 nm at each pH during the titrations.…”
Section: Methodsmentioning
confidence: 99%
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