A single mutation disrupts the pH-dependent dimerization of glycinamide ribonucleotide transformylase.
Monomeric GART reversibly associates into a dimeric form as a function of decreasing solution pH. The transition is consistent with a three-proton transfer reaction with an apparent pKa near 7. We now report that a single mutation, which replaces a glutamic acid at position 70 in the dimer interface with alanine (E70A), disrupts the pH-dependent dimerization of GART based on dynamic light scattering and gel filtration studies. A comparison of data obtained from UV-absorbance difference spectroscopy for both the wild-type and mutant forms of GART indicates that a tyrosine residue(s) undergoes a change in solvent exposure over the pH range 6.55 to 8.19. A conformational change in tertiary structure that accompanies dimerization accounts for 60% of the observed optical difference, while the remaining 40% can be attributed to a pH-dependent process unrelated to dimerization. In addition, fluorescence studies of the mutant protein indicate that a pH-dependent change in tryptophan fluorescence exhibited by the wild-type protein is unrelated to quaternary structural changes and is likely a result of simple fluorescence quenching by nearby protonated histidine side-chains. Taken together, our results indicate that a single amino acid change at the dimer interface is sufficient to interrupt the highly specific, pH-dependent assembly reaction of GART, although pH-dependent conformational changes present in the wild-type protein also occur in E70A GART. This work is a first application of structure-based site-directed mutagenesis to the analysis of this pH-dependent assembly reaction.
Mullen
C A
CA
Department of Chemistry and Biochemistry, University of California, San Diego, CA 92093-0359, USA.
Jennings
P A
PA
D019943:Amino Acid Substitution; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D019877:Hydroxymethyl and Formyl Transferases / Q000737:chemistry* / Q000235:genetics*; D008027:Light; D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D051026:Phosphoribosylglycinamide Formyltransferase; D017354:Point Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase