Dissociation of the tetrameric phosphoglycerate mutase from yeast by a mutation in the subunit contact region.
Phosphoglycerate mutases from different sources exhibit a variety of quaternary structures (tetramer, dimer and monomer). To perturb the tetrameric structure of yeast phosphoglycerate mutase we have prepared a mutant enzyme in which Lys-168 in the subunit-contact region has been replaced by proline. The K168P mutant enzyme undergoes dissociation to dimers at low concentrations; thus on lowering the concentration from 200 micrograms/ml to 5 micrograms/ml the proportion of tetramer falls from 85% to 53%. The tetrameric structure of the wild-type enzyme remains intact over this range of concentrations. The mutant enzyme has similar kinetic properties to the wild-type enzyme, with kcat. being reduced by 26%. Far-u.v. c.d. studies show that there has been a small loss of helical structure in the mutant. Compared with wild-type enzyme, the K168P mutant enzyme is slightly less stable towards proteolysis by trypsin, but significantly less stable towards denaturation by guanidinium chloride, with the midpoint concentration of guanidinium chloride some 50% lower. After denaturation, the mutant enzyme could regain activity and quaternary structure when the guanidinium chloride concentration was lowered to 0.05 M. The properties of the mutant enzyme are discussed in terms of other dimeric phosphoglycerate and bisphosphoglycerate mutases which contain proline at position 168.
White
M F
MF
Department of Biochemistry, University of Edinburgh, Scotland, U.K.
Fothergill-Gilmore
L A
LA
Kelly
S M
SM
Price
N C
NC
D002942:Circular Dichroism; D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010736:Phosphoglycerate Mutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011392:Proline; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D006146:Guanidines; D046911:Macromolecular Substances; D011392:Proline; D010736:Phosphoglycerate Mutase; D019791:Guanidine; D008239:Lysine