Human Phosphoglycerate Kinase: II. STRUCTURE OF A VARIANT ENZYME

Abstract

Abstract A human phosphoglycerate kinase variant, associated with faster anodal electrophoretic mobility in starch gel, was purified and crystallized from red blood cells of subjects in New Guinea. An apparently identical variant enzyme was also purified from a male in the Samoa Islands. The variant enzyme had the same molecular weight, 50,000, and the same specific enzyme activity, 700 units per mg, as the wild type enzyme. The isoelectric point of the variant enzyme, determined by the isoelectric focusing method, was pH 4.7 as compared with pH 7.9 for the normal enzyme. Although active variant enzyme moved toward the anode much faster than the normal enzyme in starch gel electrophoresis using a Tris-citrate buffer system at pH 7.5, no significant difference was found in electrophoretic mobility of the enzymes in other buffer systems without citrate. Lyophilized preparations of the normal and variant enzyme were enzymatically inactive, showed identical electrophoretic mobility in polyacrylamide gel electrophoresis using Tris-citrate buffer at pH 7.5, and had identical isoelectric points. These findings suggest that the fast electrophoretic mobility and the acidic isoelectric point of the variant enzyme are not due to intrinsic charge differences caused by the amino acid substitution, but due to a secondary alteration of the protein charge presumably induced by association with citrate. In fact, an electrically neutral amino acid substitution, from threonine in the normal enzyme to asparagine in the variant enzyme, was suggested by peptide mapping of their tryptic digests. The phosphoglycerate kinase variant found in a male from the Samoa Islands was identical with the variant enzyme found in New Guinea.