Potato tuber isoapyrases: substrate specificity, affinity labeling, and proteolytic susceptibility

Area of Science:

• Biochemistry
• Enzymology

Background:

• Apyrase (ATP-diphosphohydrolase) catalyzes the hydrolysis of nucleoside di- and triphosphates.
• Potato tubers contain isoapyrases with varying ATPase/ADPase ratios, influencing their enzymatic properties.

Purpose of the Study:

• To investigate substrate specificity and kinetic parameters (K(m), k(cat)) of homogeneous potato isoapyrases.
• To compare the substrate hydrolysis profiles of Desiree (low ratio) and Pimpernel (high ratio) isoapyrases.
• To explore differences between potato apyrases and animal enzymes using nucleotide analogs.

Main Methods:

• Purification of homogeneous isoapyrases from two potato clonal varieties.
• Kinetic analysis using deoxyribonucleotides and fluorescent ATP/ADP analogs.
• Affinity labeling studies with azido-nucleotides and FSBA.
• Proteolytic susceptibility assays with trypsin, chymotrypsin, and Glu-C.

Main Results:

• Both isoapyrases displayed broad specificity for various deoxyribonucleotides and fluorescent nucleotide analogs.
• Pimpernel apyrase exhibited higher hydrolytic activity on triphosphorylated compounds.
• Introduction of bulky groups on nucleotides reduced catalytic rate (k(cat)) but not substrate binding affinity (K(m)).
• Potato apyrases were insensitive to affinity labeling agents like azido-nucleotides and FSBA, unlike some animal enzymes.
• Proteolytic digestion patterns differed between the two potato isoapyrases.

Conclusions:

• Potato isoapyrases possess broad substrate specificity and distinct kinetic properties.
• Enzyme structure accommodates bulky nucleotide analogs without significant steric hindrance at the binding site.
• Differences in sensitivity to chemical modification and proteolytic cleavage highlight structural variations between potato isoapyrases.