Engineering the allosteric properties of archaeal non-phosphorylating glyceraldehyde-3-phosphate dehydrogenases.
The archaeal non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN, EC 1.2.1.9) is a highly allosteric enzyme activated by glucose 1-phosphate (Glc1P). Recent kinetic analyses of two GAPN homologs from Sulfolobales show different allosteric behaviors toward the substrate glyceraldehyde-3-phosphate (GAP) and the allosteric effector Glc1P. In GAPN from Sulfolobus tokodaii (Sto-GAPN), Glc1P-induced activation follows an increase in affinity for GAP rather than an increase in maximum velocity, whereas in GAPN from Sulfolobus solfataricus (Sso-GAPN), Glc1P-induced activation follows an increase in maximum velocity rather than in affinity for GAP. To explore the molecular basis of this difference between Sto-GAPN and Sso-GAPN, we generated 14 mutants and 2 chimeras. The analyses of chimeric GAPNs generated from regions of Sto-GAPN and Sso-GAPN indicated that a 57-residue module located in the subunit interface was clearly involved in their allosteric behavior. Among the point mutations in this modular region, the Y139R variant of Sto-GAPN no longer displayed a sigmoidal K-type-like allostery, but instead had apparent V-type allostery similar to that of Sso-GAPN, suggesting that the residue located in the center of the homotetramer critically contributes to the allosteric behavior.
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; Present address: Molecular and Computational Biology, University of Southern California, 1050 Childs Way, Los Angeles, CA 90089, USA.
Fumiaki
F
Ito
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Masayuki
M
Miyake
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Shinya
S
Fushinobu
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Shugo
S
Nakamura
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Kentaro
K
Shimizu
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Electronic address: atwakag@mail.ecc.u-tokyo.ac.jp.
Takayoshi
T
Wakagi
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005958:Glucosephosphates / Q000737:chemistry / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016944:Sulfolobus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D048229:Sulfolobus solfataricus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D005958:Glucosephosphates; D007527:Isoenzymes; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C031590:glucose-1-phosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
Allosteric enzyme; Archaea; K-type/V-type; Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN); Positive cooperativity