Proton magnetic resonance studies of ribonuclease T 1. Assignment of histidine-40 peak and analysis of the active site

Abstract

Nuclear magnetic resonances of the C-2 protons of the three histidine residues in ribonuclease T 1 have been studied at 360 MHz as a function of pH to discuss the structure of the active site. Comparison of the order of deuterium exchange of the histidine peaks with tritium incorporation rates into individual histidines of the enzyme leads to the unambigous assignment of one of the C-2 proton peaks to histidne-40. It has been concluded that histidine-40 is in the active site, interacting with a charged group of pK 4.1, which is replaced by the phosphate group of guanosine-3′-monophosphate in the enzyme-inhibitor complex. Histidine-92 is most likely a binding site for the complex, where the existence of a hydrogen bond between N-7 of the inhibitor and the ring NH proton of the histidine is suggested on the basis of NMR data.