Monitoring Enzyme Reactions by 1H NMR

The protocol for monitoring the reaction of N1 or N2 with 2,3-butadienoate (allene), 2-butynoate, and propiolate by ¹H NMR spectroscopy followed that described elsewhere.^{25 (link),26 (link)} Accordingly, an amount of each compound (4 mg) was added to 100 mM Na₂HPO₄ buffer (pH ~ 9.2) containing 30 μL of dimethyl sulfoxide-d₆ (DMSO-d₆) (final total volume of 600 μL). The final pH of the solution was adjusted to 8.0. Subsequently, an aliquot of N1 or N2 was added to the mixture such that the final amount of protein was 0.5 mg. For overnight NMR runs (i.e., 18 h) product formation was determined using a Bruker AVANCE III 500 MHz spectrometer (Billerica, MA). For shorter NMR runs, product formation was monitored every 3 min for 10 intervals on a Varian DirectDrive 600 MHz spectrometer (Palo Alto, CA). The initial spectrum was recorded 3 min after mixing. DMSO-d₆ was used as the lock signal and for standardization of the chemical shifts (at 2.49 ppm). The chemical shifts for the products are reported elsewhere.^{7 (link),26 (link),27 (link)} The approximate amount of product in the mixture was determined by integration, as reported elsewhere.^{26 (link)} NMR signals were analyzed using the software program SpinWorks 3.1.6 (Copyright © 2009 Kirk Marat, University of Manitoba).