Abstract
In a previous phylogenetic study of the family of pyruvate kinase, we found one cluster with Glu117 and another with Lys117. Those sequences with Glu117 have Thr113 and are K+-dependent, whereas those with Lys117 have Leu113 and are K+-independent. The carbonyl oxygen of Thr113 is one of the residues that coordinate K+ in the active site. Even though the side chain of Thr113 does not participate in binding K+, the strict co-evolution between position 117 and 113 suggests that T113 may be the result of the evolutionary pressure to maintain the selectivity of pyruvate kinase activity for K+. Thus, we explored if the replacement of Thr113 by Leu alters the characteristics of the K+ binding site. We found that the polarity of the residue 113 is central in the partition of K+ into its site and that the substitution of Thr for Leu changes the ion selectivity for the monovalent cation with minor changes in the binding of the substrates. Therefore, Thr113 is instrumental in the selectivity of pyruvate kinase for K+.
Highlights
Rabbit muscle pyruvate kinase (WT-RMPK) was the first enzyme for which an absolute requirement for K+ was documented [1]
In confirmation of previous results [2], we observed that WT-RMPK reached maximal activation in the presence of K+ followed by NH4+, Rb+ and Cs+ (Figure 2A); in T113L-RMPK, maximal activation followed the order Rb+ > NH4+ > K+ > Cs+ (Figure 2B)
It is noted that the Km or K0.5 for all cations studied, except for Cs+, were higher in the mutant than in the WT-RMPK; the relative increase in Km was different for each cation, indicating that T113 may participate in the selectivity of pyruvate kinases (PKs) for a monovalent cation
Summary
Rabbit muscle pyruvate kinase (WT-RMPK) was the first enzyme for which an absolute requirement for K+ was documented [1]. It has been shown that K+ changes the kinetic mechanism of WT-RMPK, inducing the closure of the active site and promoting a rearrangement of the residues involved in the binding of ADP-Mg [8]. The crystal structures of the enzyme show that K+ lies in a site formed by the carbonyl oxygen of T113, Oγ of S76, Oδ1 of N74 and Oδ1 of D112 [9,10] (Figure 1), a water molecule and a phosphate oxygen of either phosphoenolpyruvate (PEP) [11] or γ-phosphate of ATP [12].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
