title: Kinetic consequences of site-specific mutation of Glu-239----Gln in E. coli aspartate transcarbamylase: comparison with catalytic subunits and Phe-240 mutant enzyme.
abstract: The kinetic characteristics of E. coli aspartate transcarbamylase, altered by site-specific mutagenesis of Glu-239----Gln, have been determined by equilibrium isotope-exchange kinetics and compared to the wild-type system. In wild-type enzyme, residue Glu-239 helps to stabilize the T-state structure by multiple bonding interactions with Tyr-165 and Lys-164 across the c1-c4 subunit interface; upon conversion to the R-state, these bonds are re-formed within c-chains. Catalysis of both the [14C]Asp in equilibrium C-Asp and [32P]ATP in equilibrium Pi exchanges by mutant enzyme occurs at rates comparable to those for wild-type enzyme. Saturation with different reactant/product pairs produced kinetic patterns consistent with strongly preferred order binding of carbamyl-P prior to Asp and carbamyl-Asp release before Pi. The kinetics for the Gln-239 mutant enzyme resemble those observed for catalytic subunits (c3), namely a R-state enzyme (Hill coefficient nH = 1.0) and Km (Asp) approximately equal to 6 mM. The Glu-239----Gln mutation appears to destablize both the T- and R-states, whereas the Tyr-240----Phe mutation destablizes only the T-state.
authors.affiliation: Department of Molecular and Cell Biology, P.M. Althouse Laboratory, Pennsylvania State University, University Park 16802.
authors.forename: Y
authors.initials: Y
authors.lastname: Hsuanyu
authors.forename: F C
authors.initials: FC
authors.lastname: Wedler
authors.forename: S A
authors.initials: SA
authors.lastname: Middleton
authors.forename: E R
authors.initials: ER
authors.lastname: Kantrowitz
mesh_terms: D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology; D006860:Hydrogen Bonding; D007700:Kinetics; D013329:Structure-Activity Relationship
chemical_list: D001221:Aspartate Carbamoyltransferase