Heat inactivation of thermolabile polygalacturonase down to single molecule level. Systematic investigation and molecular modeling

Abstract

Polygalacturonase is an important enzyme related to quality of fruits and vegetables that it is usually inactivated by heat. This research aimed to determine the structural changes associated with the thermal inactivation of thermolabile polygalacturonase (PG2), which were studied using molecular modeling and by measuring the free sulfhydryl and intrinsic fluorescence changes. The inactivation followed a first-order kinetics during 5 min of heating (50–80 ◦C). The in silico investigation at single molecule level revealed that the temperature increase up to 80 ◦C affected the overall conformation of the catalytic site. When compared to the native enzyme, important changes of the surface available to the solvent of two catalytic amino acids, Asp202 and His223, were noticed at 70 ◦C (-27.7% and +108.6%, respectively). Further temperature increase up to 80 ◦C disrupted the hydrogen bonds connecting the amino acids within the catalytic site (His223–Asp201) and between the catalytic and binding site (Asp201-Lys258). Intrinsic fluorescence changes revealed perturbation of the tertiary structure and were in line with an all-or-none process. The evolution of free sulfhydryl indicated reaction of the Cys109 residue. The most important event in the thermal inactivation of PG2 is likely the narrowing of the access to the catalytic site.