NMR titration studies of histidine 57 and the [methylene-13C]PMS group in the phenylmethanesulfonyl (PMS) derivative of Streptomyces erythraeus trypsin.
1H and 13C NMR titrations were performed on PMS-St. trypsin derived by PMSF modification of Ser 195 in Streptomyces erythraeus trypsin, which is devoid of auto-catalytic degradation activity at pH approximately 8. NMR titration of the imidazole C2 proton showed that His 57 had the pKa value of 6.9 in both native St. trypsin and PMS-St. trypsin, suggesting that the adjacent hydroxyl group of Ser 195 had no effect or a very weak effect on the acid-base properties of the imidazole ring in the catalytic triad. A small change in the chemical shift of the isotopically enriched methylene carbon in the PMS moiety of [methylene-13C]PMS-St. trypsin was observed between pH 6 and 8. The titration curve had an inflexion at pH 6.9 and the mode of transition was apparently sigmoidal, although a Hill coefficient of more than unity was suggested. It is thus likely that His 57 is responsible for this transition. Based on these results, the role of His 57 in the catalytic triad of the active site in serine protease is discussed.
D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D006639:Histidine / Q000032:analysis*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010664:Phenylmethylsulfonyl Fluoride; D013302:Streptomyces / Q000201:enzymology*; D013450:Sulfones; D013997:Time Factors; D014357:Trypsin
D000596:Amino Acids; D013450:Sulfones; D006639:Histidine; D010664:Phenylmethylsulfonyl Fluoride; D014357:Trypsin