A study of protons associated with low-barrier hydrogen bonds in 2-amino-3-ketobutyrate-CoA ligase (AKB-ligase, EC 2.3.1.29) by NMR is reported. Three resonances are observed in the range of delta H = 15-20 ppm when the NMR spectrum of AKB-ligase is recorded at 600 MHz. These low-barrier hydrogen bonds are associated respectively with a side chain proton, the PLP pyridinium ring nitrogen proton, and the PLP Schiff base proton at the active site of the ligase. The pyridinium proton has been assigned a chemical shift of 19.10 ppm and the Schiff base proton 14.90 ppm. The third low-barrier hydrogen bond associated proton resonating at 16.20 ppm is assigned to a proton of a side chain group. All three resonances disappear when pyridoxal phosphate is removed from the ligase. Consistent with NOE coupling, the side chain group proton should be close to the proton of the Schiff base nitrogen of the pyridoxal 5'-phosphate. The effects of temperature, pH, substrate, and NOE on the three resonances are also studied, in order to assign the protons. The three low-barrier hydrogen bonds described in this report may serve to anchor the cofactor in the active site of 2-amino-3-ketobutyrate-CoA ligase.