NMR spectroscopy studies of the action pattern of tomato pectinesterase: generation of block structure in pectin by a multiple-attack mechanism

The enzymatic de-esterification of highly exterified poly(d-galacturonate) (degree of esterification, d.e. ≈ 0.92, dpn ≈ 15) was monitored by 1H NMR spectroscopy. The enzymatic reaction resulted in a high content of homogeneous triads (GGG and EEE) demonstrating the production of a sequential structure. At high d.e. values, the enzymatic activity increased in proportion to the content of GE-diads. A multiple-attack mechanism was indicated by a relatively slow decrease of single Gʹs (EGE) in the sequential structure. Production of EGG-triads in great preference to GGE-triads pointed to a degree of multiple-attack greater than the effective number average block length of E-residues, ≈ 7–8, and, interestingly, the residue at the reducing end was de-esterified faster than that at the non-reducing end. These findings support the assumption that the enzyme attacks in alternating sequences (-GE-) and de-esterifies linearly preferentially towards the reducing end. Simulation-computed results are reported to demonstrate the difference between multiple-attack and multiple-chain mechanisms.