The K+-Dependent and -Independent Pyruvate Kinases Acquire the Active Conformation by Different Mechanisms

Abstract

Eukarya pyruvate kinases possess glutamate at position 117 (numbering of rabbit muscle enzyme), whereas bacteria have either glutamate or lysine. Those with E117 are K+-dependent, whereas those with K117 are K+-independent. In a phylogenetic tree, 80% of the sequences with E117 are occupied by T113/K114/T120 and 77% of those with K117 possess L113/Q114/(L,I,V)120. This work aims to understand these residues’ contribution to the K+-independent pyruvate kinases using the K+-dependent rabbit muscle enzyme. Residues 117 and 120 are crucial in the differences between the K+-dependent and -independent mutants. K+-independent activity increased with L113 and Q114 to K117, but L120 induced structural differences that inactivated the enzyme. T120 appears to be key in folding the protein and closure of the lid of the active site to acquire its active conformation in the K+-dependent enzymes. E117K mutant was K+-independent and the enzyme acquired the active conformation by a different mechanism. In the K+-independent apoenzyme of Mycobacterium tuberculosis, K72 (K117) flips out of the active site; in the holoenzyme, K72 faces toward the active site bridging the substrates through water molecules. The results provide evidence that two different mechanisms have evolved for the catalysis of this reaction.

Effect of Aggregation and Molecular Size on the Ice Nucleation Efficiency of Proteins

“… 3D structures of proteins investigated in this study from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) ( https://www.rcsb.org/ ; Berman et al 42 ) of (a) RuBisCO (PDB ID 8RUC ; Andersson 43 ), (b) pyruvate kinase (PDB ID 7R6Y ; Ramirez-Silva et al. 44 ), (c) alkaline phosphatase (PDB ID 1ELX ; Stec et al 45 ), (d) lipase (PDB ID 1AKN ; Wang et al 46 ), and (e) insulin (PDB ID 4M4L ; Frankaer et al 47 ). Each protein is shown from a front view.…”

“…Ice Nucleation. Ice nucleation measurements were conducted using a custom-built freezing apparatus optimized from Fornea et al 48 to contain a freezing array, enabling the measurement of ice nucleation in 16 samples simultane- 42 ) of (a) RuBisCO (PDB ID 8RUC; Andersson 43 ), (b) pyruvate kinase (PDB ID 7R6Y; Ramirez-Silva et al 44 ), (c) alkaline phosphatase (PDB ID 1ELX; Stec et al 45 ), (d) lipase (PDB ID 1AKN; Wang et al 46 ), and (e) insulin (PDB ID 4M4L; Frankaer et al A three-point temperature calibration was performed to verify accuracy of the recorded temperature for our experiments using the established melting temperatures of ndodecane, n-undecane, and n-decane. 15,33,48 Individual calibrations were used for each droplet compartment within the array as a result of temperature gradients across the cooling stage.…”