A Point Mutation Increasing the Stability of Human Phosphoglucose Isomerase

Abstract

Abstract A method has been developed for the rapid isolation of human phosphoglucose isomerase by substrate-induced elution from phosphocellulose. The high degree of selectivity of the elution provides homogeneous, crystalline enzyme from erythrocytes with a 30,000-fold purification and an overall yield of approximately 70%. A genetic variant form of the enzyme (Singh variant) has also been isolated from erythrocytes of an individual heterozygous for this rare allele. The physical, chemical, and catalytic properties of the normal wild type phosphoglucose isomerase have been compared with those of the variant protein. Sedimentation equilibrium ultracentrifugation showed that both the wild type and variant enzymes are of identical molecular weights (wild type, 132,000 ± 2,000; Singh variant, 131,000 ± 3,000) and are dimers composed of identical subunits. Although the wild type isomerase electrofocused as a single component with an isoelectric pH of 9.25, the enzyme isolated from the Singh variant erythrocytes was resolved into the wild type homodimer (pI 9.25), the mixed heterodimer (pI 9.40), and the variant homodimer (pI 9.57). The wild type and variant homodimers were S-carboxymethylated, and the lysine residues carbamylated with potassium cyanate. Following tryptic digestion, peptide maps revealed a single altered peptide from the variant isomerase. The amino acid substitution in the variant is believed to be an acidic → neutral replacement. This substitution has no effect on the catalytic properties of the enzyme. However, the variant homodimer is more stable toward heat or treatment with a variety of denaturants such as guanidine hydrochloride, urea, or sodium dodecyl sulfate than is than is the wild type enzyme. Thus, while the point mutation does not affect the binding of substrates or inhibitors, it has a readily measurable effect on the over-all stability of the protein. The increased stability of the variant may account for the increased levels of phosphoglucose isomerase activity measured in blood from individuals heterozygous for this genetic phenotype.