Electrostatic interactions play a significant role in the affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase for Mn2+.
Phosphoenolpyruvate (PEP) carboxykinases catalyse the reversible formation of oxaloacetate (OAA) and ATP (or GTP) from PEP, ADP (or GDP) and CO(2). They are activated by Mn(2+), a metal ion that coordinates to the protein through the epsilon-amino group of a lysine residue, the N(epsilon-2)-imidazole of a histidine residue, and the carboxylate from an aspartic acid residue. Neutrality in the epsilon-amino group of Lys213 of Saccharomyces cerevisiae PEP carboxykinase is expected to be favoured by the vicinity of ionised Lys212. Glu272 and Glu284, located close to Lys212, should, in turn, electrostatically stabilise its positive charge and hence assist in keeping the epsilon-amino group of Lys213 in a neutral state. The mutations Glu272Gln, Glu284Gln, and Lys212Met increased the activation constant for Mn(2+) in the main reaction of the enzyme up to seven-fold. The control mutation Lys213Gln increased this constant by ten-fold, as opposed to control mutation Lys212Arg, which did not affect the Mn(2+) affinity of the enzyme. These observations indicate a role for Glu272, Glu284, and Lys212 in assisting Lys213 to properly bind Mn(2+). In an unexpected result, the mutations Glu284Gln, Lys212Met and Lys213Gln changed the nucleotide-independent OAA decarboxylase activity of S. cerevisiae PEP carboxykinase into an ADP-requiring activity, implying an effect on the OAA binding characteristics of PEP carboxykinase.
Sepúlveda
Carolina
C
Facultad de Química y Biología, Universidad de Santiago de Chile, Av. B. O'Higgins 3363, Santiago 9170022, Chile.
Poch
Andrea
A
Espinoza
Rodrigo
R
Cardemil
Emilio
E
D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D049109:Cell Proliferation; D002942:Circular Dichroism; D007700:Kinetics; D008345:Manganese / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D062907:Oxaloacetic Acid / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D055672:Static Electricity
D062907:Oxaloacetic Acid; D008345:Manganese; D000244:Adenosine Diphosphate; D002262:Carboxy-Lyases