Purification of isopentenyl pyrophosphate isomerase and geranylgeranyl pyrophosphate synthase from Capsicum chromoplasts by affinity chromatography

Abstract

A procedure based on affinity chromatography was used for the purification to homogeneity of isopentenyl pyrophosphate isomerase and geranylgeranyl pyrophosphate synthase from Capsicum chromoplasts. Isopentenyl pyrophosphate isomerase is a monomeric protein and has a molecular weight of 33 500 ± 500. The Michaelis constant for isopentenyl pyrophosphate was 6 μM. Geranylgeranyl pyrophosphate synthase has a native molecular weight of 74 000 ± 2000 resulting from the association of two apparently identical subunits having a molecular weight of 37000 ± 1000. The dimeric structure of geranylgeranyl pyrophosphate synthase was confirmed using a photolabile analog of geranyl pyrophosphate (2-diazo-3-trifluoropropionyloxygeranyl pyrophosphate). Geranylgeranyl pyrophosphate synthase catalyzed the prenyl transfer reaction isopentenyl pyrophosphate ( K m = 3 μM) and either dmiethylallyl pyrophosphate ( K m = 0.95 μM), pyrophosphate ( K m = 1 μM) or farnesyl pyrophosphate ( K m = 1.2 μM) as aliylic partners. The three prenyl transfer reactions were inhibited when the enzyme was irradiated with the photolabile analog of geranyl pyrophosphate.