THE CONFORMATION OF COENZYME-A BOUND TO CHLORAMPHENICOL ACETYLTRANSFERASE DETERMINED BY TRANSFERRED NOE EXPERIMENTS

The conformation of coenzyme A bound to chloramphenicol acetyltransfer ase has been studied in solution by NMR methods. Transferred nuclear O verhauser enhancement (NOE) and rotating frame NOE (ROE) experiments w ere used to determine the conformation of the bound coenzyme. Experime nts were carried out at five mixing times and two temperatures, and wi th normal and perdeuterated enzyme, to ensure (1) that the fast exchan ge condition was satisfied and (2) that the results were not complicat ed by spin diffusion involving enzyme protons. The data were analysed using a general approach involving combined exchange and relaxation ma trices. For the binary complex of coenzyme A (CoA) and enzyme, the con formation of CoA was calculated by using distance constraints derived from the intensities of 71 NOE and 33 ROE cross-peaks between coenzyme protons. The conformation of the adenosine moiety of CoA in the struc ture deduced by NMR is very close to that seen in the crystal structur e of this complex, while the pantetheine moiety is clearly less extend ed. Essentially the same conformation was obtained whether or not the calculations included the protein (with appropriate intermolecular ene rgy terms). The difference between the NMR and X-ray structures is int erpreted in terms of the existence of two conformations of the CoA-enz yme complex. Support for this model comes from measurements of the coe nzyme dissociation rate constant; NMR (lineshape analysis and transfer red NOE experiments) gives estimates of k(off) similar to 3700 s(-1) a t 298 K and similar to 500 s(-1) at 280 K, both significantly greater than estimates by fluorescence stopped-flow measurements. For the tern ary complex of CoA, chloramphenicol and enzyme, 71 NOE cross-peaks bet ween protons of coenzyme A and a further ten cross-peaks between proto ns of coenzyme A and chloramphenicol were measured. Starting with a mo del derived from the crystal structures of the two binary complexes (i n the absence of crystallographic data for the ternary complex) the co nformations and relative positions of the two ligands were refined usi ng the distance constraints derived from these NOEs. The conformation of the adenosine part of CoA is the same as in the binary complex, whi le the pantetheine arm is more extended and approaches close to the bo und chloramphenicol molecule. The model of the ternary complex is disc ussed in terms of the information available on the mechanism of the en zyme. (C) 1996 Academic Press Limited