Use of 1H-15N heteronuclear multiple-quantum coherence NMR spectroscopy to study the active site of aspartate aminotransferase.
Aspartate aminotransferase from Escherichia coli, an 88 kDa enzyme, was uniformly and selectively enriched with 15N and was studied by heteronuclear multiple-quantum coherence NMR spectroscopy in H2O. Good resolution was obtained for the downfield region (above 9.5 ppm chemical shift in the 1H dimension) for NH protons in the amide, indole, imidazole, and guanidinium group regions and several resonances were tentatively assigned. Two downfield resonances, at 12.6 and 11.36 ppm, appear to belong to oxygen- or sulfur-bound protons. The most downfield amide resonance at 11.78 ppm was assigned to the active site cysteine 192 whose peptide proton is 2.9 A away from the negatively charged carboxyl group of aspartate 199. Large downfield shifts (up to 1.15 ppm) of the indole NH resonance of the active site tryptophan 140 were observed upon binding of dicarboxylic inhibitors to the pyridoxal 5'-phosphate (PLP) form and of inorganic dianions to the pyridoxamine 5'-phosphate (PMP) form of the enzyme. We discuss these striking differences in the light of the available crystallographic data. Active sites of proteins, as well as specific inhibitory molecules, often contain negatively charged groups. These may be able to form hydrogen-bonds to NH 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 observations, should be widely applicable.
Mollova
E T
ET
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011, USA.
Metzler
D E
DE
Kintanar
A
A
Kagamiyama
H
H
Hayashi
H
H
Hirotsu
K
K
Miyahara
I
I
D001219:Aspartate Aminotransferases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008970:Molecular Weight; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011733:Pyridoxamine / Q000031:analogs & derivatives / Q000378:metabolism
D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D001219:Aspartate Aminotransferases; C010627:pyridoxamine phosphate