EVIDENCE FOR DOMAIN-STRUCTURES OF THE TRIFUNCTIONAL PROTEIN AND THE TETRAFUNCTIONAL PROTEIN ACTING IN GLYOXYSOMAL FATTY-ACID BETA-OXIDATION

In plant glyoxysomes, an enzyme activity responsible for a particular step in the fatty acid beta-oxidation is located on more than one prot ein species. Various monofunctional enzymes and two forms of a multifu nctional protein are involved in the degradation of cis-unsaturated fa tty acids. Delta(3),Delta(2)-Enoyl-CoA isomerase activity, previously found to be located on a monofunctional dimeric protein, is attributab le to one form of the monomeric multifunctional protein (MFP). The pre sence or absence of isomerase activity allows us to differentiate betw een the tetrafunctional 76.5-kDa isoform (MFP II) and the trifunctiona l 74-kDa isoform (MFP I) in cucumber (Cucumis sativus) cotyledons. Bot h MFP I and MFP II exhibited blocked N-terminal structures. MFP I and MFP II are distinguishable from each other by their susceptibility to limited proteolysis. A series of examples is presented describing the preparation of enzymically active proteolytic fragments. We demonstrat e that both forms of the monomeric MFP are composed of domains separab le from each other without loss of activity. By fragmentation of NFP I and subsequent chromatography, a 60-kDa peptide was purified retainin g hydratase and epimerase activity but lacking dehydrogenase activity. In addition, a highly positively charged fragment was observed carryi ng solely dehydrogenase activity. From MFP II, a 36-kDa fragment with hydratase activity was characterized. An enzymically inactive 46-kDa f ragment was prepared from MFP II and sequenced at its unblocked N-term inus.