Zymomonas mobilis pyruvate kinase: Rise of the unstable zombies.

Abstract

Pyruvate kinase (PYK) is the final enzyme of glycolysis and is present in all domains of life. Mutations in PYK isozymes have a range of outcomes. Single amino acid changes in the human liver isoform (hLPYK) “tune” the Km for phosphoenolpyruvate by ∼200-fold relative wild-type; very few changes (∼10%) abolished enzyme activity. In contrast, most single substitutions in the distantly-related, Zymomonas mobilis PYK (ZmPYK) either had Km values like wild-type (∼50%) or abolished enzyme activity (∼47%). To investigate the molecular basis for substitution sensitivity, we determined the crystal structure of ZmPYK. Although ZmPYK elutes from a size exclusion column as a dimer, it crystalized as a tetramer, but with a less-extensive protein-protein interface than hLPYK. The ZmPYK structure also showed a phosphate bound in the pocket analogous to hLPYK's fructose-1,6-bisphosphate allosteric site. Thermal denaturation and activity assays suggest that phosphate plays a structural role rather than an allosteric role. By adding phosphate and modifying the protein preparation, the activity for previously-dead ZmPYK variants could be recovered (“zombies”). Kapp-PEP values for ZmPYK zombies fell within a narrow range, near the wild-type values. To assess stability of ZmPYK, we performed thermal denaturation experiments, which was irreversible for all isozymes. ZmPYK had a reproducibly lower Tm-app than hLPYK, and zombie ZmPYK variants had reproducibly lower Tm-app values than wild-type-like ZmPYK variants. Taken together, these results suggest that the amino acid changes of the zombie ZmPYK variants may decrease protein stability. The results leave open the question as to why Kapp-PEP for ZmPYK is less tunable than for hLPYK. Future studies will focus on the interplay of tuneability and allosteric regulation, since hLPYK is allosterically regulated by several effectors, whereas neither phosphate nor other potential effectors have shown allosteric regulation for ZmPYK.