Proton NMR studies of a large protein. pH, substrate titrations, and NOESY experiments with perdeuterated yeast phosphoglycerate kinase containing [1H]histidine residues.
Fully deuterated yeast phosphoglycerate kinase ([2H]PGK) was prepared biosynthetically with only histidine side chains of normal (1H) isotopic composition. The 1H NMR spectrum of this enzyme ([1H]His[2H]PGK) showed that the histidine side chains are clearly visible as sharp signals. Thus detailed structural studies by 1H NMR became feasible with isotope-hybrid phosphoglycerate kinase which is otherwise too large (M(r) approximately 46,000) for conventional 1H NMR studies. Proton signals of bound substrates were visible in the 1H NMR spectrum even with a substrate-to-enzyme ratio of less than 1/2 (mol/mol). The 2D NOESY spectrum of enzyme-MgdATP-glycerol 3-phosphate complex showed that, although protein concentration was very high (1.5 mM), no intraprotein cross peaks were observed other than those of intraresidue histidine NOE cross peaks. In addition, intrasubstrate NOEs and intermolecular NOEs between histidine and substrate protons were visible at a 1.5/1 substrate/enzyme (mol/mol) ratio. Paramagnetic effects of a substrate analog, Cr(III)ATP, on some of the histidine side chains indicated that the formation of the ternary enzyme-substrate complex causes large conformational changes in the enzyme.
Pappu
K M
KM
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
Serpersu
E H
EH
D000255:Adenosine Triphosphate / Q000032:analysis / Q000737:chemistry; D003903:Deuterium; D004578:Electron Spin Resonance Spectroscopy; D005994:Glycerophosphates / Q000032:analysis / Q000737:chemistry; D006639:Histidine / Q000032:analysis* / Q000737:chemistry; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010735:Phosphoglycerate Kinase / Q000032:analysis* / Q000737:chemistry; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D005994:Glycerophosphates; D006639:Histidine; D006859:Hydrogen; D000255:Adenosine Triphosphate; C029620:alpha-glycerophosphoric acid; D003903:Deuterium; D010735:Phosphoglycerate Kinase