USE OF H-1-N-15 HETERONUCLEAR MULTIPLE-QUANTUM COHERENCE NMR-SPECTROSCOPY TO STUDY THE ACTIVE-SITE OF ASPARTATE-AMINOTRANSFERASE

Aspartate aminotransferase from Escherichia coli, an 88 kDa enzyme, wa s uniformly and selectively enriched with N-15 and was studied by hete ronuclear multiple-quantum coherence NMR spectroscopy in H2O. Good res olution was obtained for the downfield region (above 9.5 ppm chemical shift in the H-1 dimension) for NH protons in the amide, indole, imida zole, and guanidinium group regions and several resonances were tentat ively assigned. Two downfield resonances, at 12.6 and 11.36 ppm, appea r to belong to oxygen- or sulfur-bound protons. The most downfield ami de resonance at 11.78 ppm was assigned to the active site cysteine 192 whose peptide proton is 2.9 Angstrom away from the negatively charged carboxyl group of aspartate 199. Large downfield shifts (up to 1.15 p pm) of the indole NH resonance of the active site tryptophan 140 were observed upon binding of dicarboxylic inhibitors to the pyridoxal 5'-p hosphate (PLP) form and of inorganic dianions to the pyridoxamine 5'-p hosphate (PMP) form of the enzyme. We discuss these striking differenc es in the light of the available crystallographic data. Active sites o f proteins, as well as specific inhibitory molecules, often contain ne gatively charged groups, These may be able to form hydrogen-bonds to N H groups and to shift the NH resonances downfield into a less crowded and therefore more readily observable region for many large proteins. Our approach, which makes use of both HMQC spectroscopy and NOE observ ations, should be widely applicable.