Interconvertible Kinetic and Physical Forms of Human Erythrocyte Pyruvate Kinase

Abstract

Abstract Human erythrocyte pyruvate kinase (EC 2.7.1.40) was isolated in forms with K0.5 s values for phosphoenolpyruvate which range from 3 x 10-5 m to greater than 10-3 m. Several activity peaks also were obtained when samples were subjected to Sephadex G-200 chromatography, sucrose gradient sedimentation, or starch gel electrophoresis. The most prevalent peak had a molecular weight of 237,000 ± 1,680 S.E. when analyzed by gel filtration chromatography and 214,000 ± 2,700 S.E. when subjected to sedimentation studies. Assuming this to be a tetrameric form, evidence was also obtained for possible monomeric, dimeric, trimeric, and pentameric forms. Enzyme maintained at pH values below 7.0 had K0.5 s values for phosphoenolpyruvate of less than 8 x 10-5 m, gave hyperbolic rate curves, and was 80% or better apparent tetramer. This form could be reversibly converted to a form which had K0.5 s values for phosphoenolpyruvate of about 2.0 x 10-4 m by aging in alkaline pH or purifying on DEAE-cellulose columns. Such preparations appeared to consist primarily of apparent trimer and could be converted back to the lower K0.5 s apparent tetrameric form by aging in solutions with pH values less than 7.0 or by incubating with fructose 1,6-diphosphate. In addition to this apparent R ⇌ T conformational pair, a form of the enzyme with a K0.5 s value for phosphoenolpyruvate greater than 7 x 10-4 m was also obtained from outdated cells. This form of the enzyme could exist in solutions with other forms, but it could not be reconverted to lower K0.5 s forms by exposure to acidic solutions. Another apparently distinct form of enzyme was found in lysates of fresh blood cells. This differed from any of those previously described in that the K0.5 s and nh values (Hill coefficient) obtained with phosphoenolpyruvate as the variable substrate were highly dependent upon the ADP concentration and in that the apparent dimeric form predominated in Sephadex and starch gel studies.