Recent Observations on the Specificity and Structural Conformation of the Polygalacturonase-Polygalacturonase Inhibiting Protein System

The interaction between fungal polygalacturonases (PGs) and plant cell wall proteins inhibitory to these pectin-degrading enzymes has been well established. By convention, these inhibitory proteins have been referred to as polygalacturonase-inhibiting proteins or PGIPs, with the typical PG/PGIP interaction being characterized by high-affinity, reversibility, and a one:one stoichiometry. Different combinations of various PG and PGIP isoforms and/or glycoforms yield differential or combination-specific degrees of enzyme inhibition, with some PG/PGIP pairings producing enzyme inhibition of as much as 99%. Recent studies in our laboratory have indicated that in some instances, given certain combinations of PGIP isoforms with certain PGs, the rate of catalysis is actually increased beyond that characteristic of the enzyme alone. Clearly, this activation or enhancement of catalytic rate constitutes a deviation from the conventional inhibitory effect attributed to this class of plant cell wall protein, suggesting the need for a re-evaluation of the conventional moniker applied to PGIPs; suggestions include Polygalacturonase-Binding Protein and Polygalacturonase-Modulating Protein. Additional revelations concerning interactions of PG, polygalacturonic acid, and PGIP arose from structural conformation studies involving amide exchange-MS, pepsin cleavage, and UV fluorescence analysis when applied to PG alone, PG/oligogalacturonide complex and PG/oligogalacturonide/PGIP complex. Indications are that the substrate oligomer contacts the surface of the PG along the cleft of the β-barrel configuration predicted for PGs by X-ray structure and site-specific mutation studies. Evidence also supports a conformational change in the PG when bound to substrate, to the effect that the hydrogen bonding in the β-pleated sheet region on the side of the PG opposite to the substrate binding site is destabilized when the enzyme is bound to the substrate. When PGIP binds the PG/substrate complex, it appears to do so on the side opposite the binding site, resulting in apparent inhibition of the conformational change induced upon binding of substrate. These results, together with previously reported data, begin to point to a location for inhibitor interaction as well as to possible mechanisms for the non-competitive inhibition often attributed to this system.