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

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-[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 ...

Detection of low-populated reaction intermediates with hyperpolarized NMR

“…Thus, enzymatic catalysis has been mostly studied at equilibrium or by investigating active enzymes together with inhibitors or inactive enzymes together with natural substrates. 1 As an alternative, cryoenzymology has been applied for the stabilization of otherwise elusive reaction intermediates in aqueous organic cryosolvents at temperatures near 250 K. 2 Physiologically more relevant enzyme studies would employ an active enzyme, physiological temperatures and aqueous solution to detect the turnover of the natural substrate. This ideal setting will, however, only be feasible at a drastically improved NMR sensitivity, if the formation of intermediates at low micromolar or even submicromolar concentrations is to be detected in fast real-time NMR experiments.…”

Steady-state carbon-13 nuclear magnetic resonance spectra of acyl .alpha.-chymotrypsin

“…There have been preliminary reports of the 13C spectrum of acetyl-a-chymotrypsin (Niu et al, 1977;Tobias et al, 1984) at low pH and/or low temperatures. Robillard et al (1974) measured the spectrum of [13C] carbonyl-enriched TV-acetyltyrosine semicarbazide in equilibrium with chymotrypsin and found no apparent change in the peak position as compared with the substrate alone.…”

Discovery of Intermediates of lacZ β-Galactosidase Catalyzed Hydrolysis Using dDNP NMR

“…Using dissolution dynamic nuclear polarization (dDNP), the sensitivity of single scan solution NMR can be increased by up to 4 orders of magnitude. 3 Conventional NMR spectroscopy has been used to study enzymatic reactions at equilibrium or for competition studies, 4 and for glycosidases, it is possible to determine whether the enzyme has a mechanism with retention or invertion of configuration. 5 Using dDNP NMR, it is possible to observe low-populated reaction intermediates, and examples include enzymatic reactions, 6 polymerization reactions, 7 and metalcatalyzed reactions.…”