Site-directed mutagenesis study of the microenvironment characteristics of Lys213 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.
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. Nature Struct. Biol. 4 (1997) 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 infrequent 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(416) in contributing to provide a low polarity microenvironment suitable to keep the epsilon-NH(2) of Lys(213) in the unprotonated form. Mutation Phe416Tyr 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). A study of the effect of pH on K(m) for Mn(2+) indicate that the affinity of recombinant wild type enzyme for the metal ion is dependent on deprotonation of a group with pK(a) of 7.1+/-0.2, compatible with the low pK(a) expected for Lys(213). This pK(a) value increases at least 1.5 pH units upon Phe416Tyr mutation, in agreement with the expected effect of an increase in the polarity of Lys(213) microenvironment. Theoretical calculations of the pK(a) of Lys(213) indicate a value of 6.5+/-0.9, and it increases to 8.2+/-1.6 upon Phe416Tyr mutation. Additionally, mutation Phe416Tyr causes a loss of 1.3 kcal mol(-1) in the affinity of the enzyme for PEP, an effect perhaps related to the close proximity of Phe(416) to Arg(70), a residue previously shown to be important for PEP binding.
Departamento de Ciencias Químicas, Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40, Santiago 33, Chile.
Alejandro
A
Yévenes
Rodrigo
R
Espinoza
Jaime A
JA
Rivas-Pardo
José M
JM
Villarreal
Fernando D
FD
González-Nilo
Emilio
E
Cardemil
D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000737:chemistry
D014443:Tyrosine; D010649:Phenylalanine; D019764:Phosphoenolpyruvate Carboxykinase (ATP); D008239:Lysine