Open Access13 C High-Resolution Nuclear Magnetic Resonance Studies of Enzyme-Substrate Reactions at Equilibrium. Substrate Strain Studies of Chymotrypsin- N -Acetyltyrosine Semicarbazide Complexes
Author(s) -
George T. Robillard,
Elliott Shaw,
Robert G. Shulman
Publication year - 1974
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.71.7.2623
Subject(s) - semicarbazide , substrate (aquarium) , chemistry , enzyme , resonance (particle physics) , hydrolysis , chymotrypsin , stereochemistry , nuclear magnetic resonance , biochemistry , organic chemistry , trypsin , biology , ecology , physics , particle physics
N -Acetyl-L-tyrosine semicarbazide is hydrolyzed by chymotrypsin (EC 3.4.21.1) toN -acetyl-L-tyrosine and semicarbazide. If a high concentration of semicarbazide is present, the equilibrium for the reaction can be shifted from hydrolysis to synthesis. UsingN -acetyl-L-[13 C]tyrosine enriched at the carboxyl carbon and high concentrations of semicarbazide hydrochloride, we have studied the enzyme-substrate complex ofN -acetyl-L-[13 C]tyrosine semicarbazide and chymotrypsin Aδ by13 C nuclear magnetic resonance. We observe no shift within the experimental accuracy of ±0.05 ppm as the fraction of substrate bound is changed from 0.17 to 0.70. Since E + S ⇄ ES is in fast exchange on the nuclear magnetic resonance time scale, it is possible to show that when the substrate is bound to the enzyme in the Michaelis complex, the13 C resonance is shifted less than 0.1 ppm, indicating that negligible substrate strain occurs in this complex at the site of enzymatic attack. These experiments demonstrate the application of nuclear magnetic resonance to the study of particular states along the reaction pathway for enzyme-substrate reactions at equilibrium.