Relevance of phenylalanine 216 in the affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase for Mn(II).
Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate and carbon dioxide, and uses Mn(2+) as the activating metal ion. Comparison with the crystalline structure of homologous Escherichia coli PEP carboxykinase [Tari et al. (1997) Nature Struct. Biol. 4, 990-994] shows that Lys(213) is one of the ligands to Mn(2+) at the enzyme active site. Coordination of Mn(2+) to a lysyl residue is not common and suggests a low pK (a) value for the epsilon-NH(2) group of Lys(213). In this work, we evaluate the role of neighboring Phe(216) in contributing to provide a low polarity microenvironment suitable to keep the epsilon-NH(2) of Lys(213) in the unprotonated form. Mutation Phe216Tyr shows that the introduction of a hydroxyl group in the lateral chain of the residue produces a substantial loss in the enzyme affinity for Mn(2+), suggesting an increase of the pK (a) of Lys(213). In agreement with this interpretation, theoretical calculations indicate an alkaline shift of 2.8 pH units in the pK (a) of the epsilon-amino group of Lys(213) upon Phe216Tyr mutation.
Yévenes
Alejandro
A
Departamento de Ciencias Químicas, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago 33, Chile.
González-Nilo
Fernando D
FD
Cardemil
Emilio
E
D019943:Amino Acid Substitution; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000737:chemistry / Q000378:metabolism*; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D029701:Saccharomyces cerevisiae Proteins; D008345:Manganese; D010649:Phenylalanine; D019764:Phosphoenolpyruvate Carboxykinase (ATP)