Substrate and inhibitor-induced conformational changes in the structurallyrelated enzymes UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and5-enolpyruvylshikimate 3-phosphate synthase (EPSPS)

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshi kimate 3-phosphate synthase (EPSPS) have both a unique three-dimensional. t opology and overall reaction mechanism in common. In the case of MurA, the substrate-free, unliganded protein exhibits an "open" conformation. Upon bi nding of substrates, the protein forms a much more tightly packed so-called "closed" form following an induced fit mechanism. In this closed form, the substrates are properly positioned for catalysis. On the basis of the stru ctural and mechanistic similarities of MurA and EPSPS, a similar conformati onal change is likely to occur in EPSPS to generate a catalytically compete nt active site. However, there is currently little experimental evidence av ailable to support the occurrence of such a conformational change in EPSPS. Using Limited tryptic digestion of MurA,(1) it could be shown that formati on of the "closed" conformation of MurA is accompanied by a marked increase of stability toward proteolytic degradation. Formation of the closed confo rmation was achieved by addition of either an excess of both Substrates or the sugar nucleotide substrate in conjunction with the antibiotic fosfomyci n. Analysis of the MurA tryptic fragments by MALDI-TOF mass spectrometry de monstrates that the protection of the protein in either case is caused by ( I) a specific shielding of regions thereby becoming less accessible as a re sult of the conformational change, and (2) an unspecific overall protection of the whole protein due to an apparently reduced flexibility of the pepti de backbone in the binary and ternary complexes. The establishment of metho ds to describe the effects of tryptic digestion on MurA under various condi tions was then extended to EPSPS. Although EPSPS was found to be much more stable toward proteolysis than MurA, the presence of shikimate 3-phosphate (S3P) and the inhibitor glyphosate led to a pronounced suppression of prote olytic degradation. When unliganded EPSPS was treated with trypsin, three o f the peptide fragments obtained could be identified by mass spectrometry. Two of these are located in a region corresponding to the "catalytic" loop in MurA which participates in the conformational change. This indicates a c onformational change in EPSPS, similar to the one observed in MurA, leading to the protection mentioned above. Corroborating evidence was obtained usi ng a conformational sensitive monoclonal antibody against EPSPS which showe d a 20-fold reduced affinity toward the protein complexed with S3P and glyp hosate as compared to the unliganded enzyme.