Nuclear magnetic resonance studies of the interaction of N-formyltryptophanate with .alpha.-chymotrypsin

Jt, Gerig; Ra, Rimerman

doi:10.1021/ja00776a044

Cited by 6 publications

(1 citation statement)

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“…However, the methods for obtaining structural information, x-ray crystallography and high-resolution nuclear magnetic resonance (NMR), are not ordinarily suited for studying true enzyme-substrate complexes because of the long times required for data accumulation, during which the substrate is converted to product. Thus, except for the technically difficult experiment combining NMR and stoppedflow kinetics (1), these methods have been applied to systems composed either of active enzyme plus inhibitors or inactive enzyme plus true substrates (2)(3)(4)(5). These results are often extrapolated to obtain information about an active enzymesubstrate complex.…”

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confidence: 99%

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

Robillard¹,

Shaw

Shulman³

1974

Proc. Natl. Acad. Sci. U.S.A.

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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-[13Cltyrosine enriched at the carboxyl carbon and high concentrations of semicarbazide hydrochloride, we have studied the enzyme-substrate complex of N-acetyl-L-[13Cltyrosine semicarbazide and chymotrypsin As by 1'3C nuclear magnetic resonance. We observe no shift within the experimental accuracy of 4-0.05 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.A large majority of enzymatic reactions proceed by a series of several distinct steps that may include, besides the enzymesubstrate complex, covalent intermediates and metastable transition states. In order to understand how an enzyme functions and to assess the forces that contribute to its catalytic efficiency, one would like to compile detailed structural and kinetic data on each state in the reaction pathway. For certain well-studied systems like chymotrypsin an abundance of kinetic data has been assembled for the interaction of true substrates with enzymes. However, the methods for obtaining structural information, x-ray crystallography and high-resolution nuclear magnetic resonance (NMR), are not ordinarily suited for studying true enzyme-substrate complexes because of the long times required for data accumulation, during which the substrate is converted to product. Thus, except for the technically difficult experiment combining NMR and stoppedflow kinetics (1), these methods have been applied to systems composed either of active enzyme plus inhibitors or inactive enzyme plus true substrates (2-5). These results are often extrapolated to obtain information about an active enzymesubstrate complex.The present report shows how it is possible to make highresolution NMR measurements on an active enzyme-substrate system by a careful choice of reaction conditions. The principle of this method is to monitor the system under equilibrium conditions while forcing the equilibrium in the direction of the substrate with high product concentrations. To allow appreciable substrate binding, this requires that the product is not a competitive inhibitor. This permits lengthy NMR measurements while maintaining a constant high substrate concentration. Jencks et al. (6) demonstrated that in the reaction of N-acetyl-L-tyrosine hydroxamic acid with chymotrypsin, the equilibrium could be ...

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

Robillard¹,

Shaw

Shulman³

1974

Proc. Natl. Acad. Sci. U.S.A.

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Effects of pD on ring‐stacking interactions between dinucleoside phosphates and tryptamine

Kolodny

Neville

1980

Biopolymers

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SynopsisComplex formation between tryptamine and mononucleotides and dinucleoside phosphates containing adenine and/or cytosine has been studied at five pD's ranging from 1.1 to 7.4 by proton magnetic resonance spectroscopy. Chemical shifts of base ring protons and the ribose anomeric proton in the nucleotides and indole ring protons in tryptamine were monitored and their changes with pD and intermolecular interactions interpreted qualitatively. Stacked complexes were found to exist at all pD's in the range studied. Complex geometries differ depending on pD. An electrostatic interaction between the tryptamine amino group and the nucleotide phosphate group contributes to complex formation above pD 4 but is not strong enough to shift the dinucleoside phosphate equilibrium towards the unstacked conformer.

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ChemInform Abstract: NMR‐UNTERSUCHUNG DER WECHSELWIRKUNG VON N‐FORMYL‐, N‐TRIFLUORACETYL‐ UND N‐ACETYL‐TRYPTOPHAN MIT ALPHA‐CHYMOTRYPSIN

Gerig¹,

Rimerman²

1972

Chemischer Informationsdienst

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Nuclear magnetic resonance studies of the interaction of N-formyltryptophanate with .alpha.-chymotrypsin

Cited by 6 publications

References 3 publications

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

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

Effects of pD on ring‐stacking interactions between dinucleoside phosphates and tryptamine

ChemInform Abstract: NMR‐UNTERSUCHUNG DER WECHSELWIRKUNG VON N‐FORMYL‐, N‐TRIFLUORACETYL‐ UND N‐ACETYL‐TRYPTOPHAN MIT ALPHA‐CHYMOTRYPSIN

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Nuclear magnetic resonance studies of the interaction of N-formyltryptophanate with .alpha.-chymotrypsin

Cited by 6 publications

References 3 publications

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

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

Effects of pD on ring‐stacking interactions between dinucleoside phosphates and tryptamine

ChemInform Abstract: NMR‐UNTERSUCHUNG DER WECHSELWIRKUNG VON N‐FORMYL‐, N‐TRIFLUORACETYL‐ UND N‐ACETYL‐TRYPTOPHAN MIT ALPHA‐CHYMOTRYPSIN

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

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