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 esterified poly(D-galacturonate) (degree of esterification, d.e. approximate to 0.92, dp(n) approximate to 15) was monitored by H-1 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 G's (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, approximate to 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. (C) 1996 Elsevier Science Ltd.