N-Acetyl-L-tyrosine semicarbazide is hydrolyzed by chymotrypsin (EC 3.4.21.1) to N-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. Using N-acetyl-L-[(13)C]tyrosine enriched at the carboxyl carbon and high concentrations of semicarbazide hydrochloride, we have studied the enzyme-substrate complex of N-acetyl-L-[(13)C]tyrosine semicarbazide and chymotrypsin A(delta) by (13)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 right arrow over left arrow 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, the (13)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.