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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1974 Jul;71(7):2623–2626. doi: 10.1073/pnas.71.7.2623

13C High-Resolution Nuclear Magnetic Resonance Studies of Enzyme-Substrate Reactions at Equilibrium. Substrate Strain Studies of Chymotrypsin-N-Acetyltyrosine Semicarbazide Complexes

George Robillard *, Elliott Shaw †,‡, R G Shulman *
PMCID: PMC388518  PMID: 4528121

Abstract

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-[13C]tyrosine enriched at the carboxyl carbon and high concentrations of semicarbazide hydrochloride, we have studied the enzyme-substrate complex of N-acetyl-L-[13C]tyrosine semicarbazide and chymotrypsin Aδ by 13C 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, the 13C 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.

Keywords: kinetic study

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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