A glutamine 67--> histidine mutation in homotetrameric R67 dihydrofolate reductase results in four mutations per single active site pore and causes substantial substrate and cofactor inhibition.
R67 dihydrofolate reductase (DHFR) is a type II DHFR produced by bacteria as a resistance mechanism to increasing clinical use of the antibacterial drug trimethoprim. Type II DHFRs are not homologous in either sequence or structure with chromosomal DHFRs. The crystal structure of R67 DHFR shows a single active site pore that spans the length of the homotetramer. Related sites (due to a 222 symmetry element at the center of the pore) are used to bind ligands, i.e. each half of the pore can accommodate either the substrate, dihydrofolate (DHF), or the cofactor, NADPH, although DHF and NADPH are bound differently. To evaluate the role of glutamine 67 (and its symmetry-related Q167, Q267 and Q367 residues which occur at the center of the active site pore), a Q67H mutation was constructed. Binary binding of dihydrofolate (DHF; monitored by isothermal titration calorimetry) displays two identical sites with a Kd value of 0.04 microM, while binding of NADPH shows two sites possessing negative cooperativity with Kd values of 0.027 and 0.62 microM. A comparison of ligand binding in Q67H versus wild-type (wt) R67 DHFR indicates both ligands bind more tightly (80-6000-fold) and DHF binding in Q67H R67 DHFR no longer displays positive cooperativity as seen in wt R67 DHFR. Ternary complex binding in the Q67H mutant indicates a total of two ligands can bind per pore. Substantial substrate and cofactor inhibition are observed during catalysis, consistent with non-productive binding of either two DHF or two NADPH molecules in Q67H R67 DHFR. Because of the symmetry-related binding sites in the active site pore, the accumulation of potentially positive mutations in R67 DHFR is limited by the balance between tighter binding of ligands (and thus potentially increased catalytic efficiency) and inhibition that arises upon tighter binding of two identical ligands at symmetry-related sites.
Park
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Biochemistry, Cell and Molecular Biology Department, University of Tennessee, Knoxville 37996-0840, USA.
Bradrick
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Howell
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D001665:Binding Sites; D002151:Calorimetry; D002384:Catalysis; D004791:Enzyme Inhibitors; D004926:Escherichia coli / Q000235:genetics; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D005973:Glutamine / Q000235:genetics*; D006632:Histamine / Q000235:genetics*; D007700:Kinetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013816:Thermodynamics
D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D005973:Glutamine; C010920:dihydrofolate; D009249:NADP; D006632:Histamine; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase