Poster: Enzymology

Abs # 238: Characterization of the regulatory role of residues 226-232 of maize PEP carboxylase

Presenter:	Mendez, Julian , jmend78@hotmail.com
Authors	Mendez, Julian  (A)   Yuan, Jiping  (A)   Sayegh, Joyce  (A)   Hartle, Laurell  (A)   Sanchez, Norma  (A)   Grover, Scott  (A)
Affiliations:	(A): California State University Los Angeles

Site-directed mutagenesis was performed on residues in a surface loop that is part of a putative glucose-6-phosphate activation site of maize phosphoenolpyruvate (PEP) carboxylase. Polar or charged amino acids between residues 226 to 232 were replaced in order to test their role in activation (by glucose-6-phosphate or glycine) or inhibition (by malate or aspartate). Substitutions leading to loss of charge at Asp 228 (D228N), Arg 231 (R231A) and Arg 232 (R232Q) decreased the enzyme’s apparent affinity for the activator glucose-6-phosphate by 4-fold, 8-fold and 48-fold, respectively. Mutation of Glu 229 increased the enzyme’s apparent affinity for glucose-6-phosphate but significantly reduced the magnitude of the activation caused by glucose-6-phosphate and, especially, glycine. Mutation of Asp 228 (D228N) reduced the enzyme’s apparent affinity for the activator glycine 17-fold. Mutation of Arg 226 (R226Q) eliminated the enzyme’s response to glycine. Mutations leading to loss of charge at Arg 226 (R226Q) and Glu 229 (E229A) decreased the enzyme’s apparent affinity for the inhibitor malate 4-fold and 44-fold, respectively. Similar changes, relative to the wild-type enzyme, were seen in the response of R226Q and E229A to the inhibitor aspartate. Mutation of Thr 227 (T227S) and Asp 228 (D228N) led, respectively, to a 6-fold and a 3-fold increase in malate sensitivity. Thus this surface loop is rich in residues that are critical determinants of the enzyme’s sensitivity to both positive and negative allosteric effectors. This work is supported by NIH MBRS-RISE grant GM61331 and NIH Biomed- PREP grant GM 64104.