Site-specific substitutions of the Tyr-165 residue in the catalytic chain of aspartate transcarbamoylase promotes a T-state preference in the holoenzyme.
The amino acid residue Tyr-165C of aspartate transcarbamoylase (EC 2.1.3.2) of Escherichia coli has been proposed to be involved in the transition from the T-state to the R-state upon binding of the bisubstrate analogue N-(phosphonacetyl)-L-aspartate. Site-specific mutagenesis has been used to substitute phenylalanine for tyrosine, thus maintaining the aromatic R-group but removing the charged hydroxyl moiety. This mutation dramatically altered the aspartate requirements for the holoenzyme but did not substantially affect the homotropic or heterotropic characteristics of the oligomer. The aspartate requirements for half-maximal saturation increased from 5.5 mM at pH 7.0 for the native holoenzyme to approximately 90 mM in the mutant enzyme. Nonetheless, estimates of the kinetic cooperativity index remained similar (Hill coefficients: Tyr-165C, n = 2.1; Phe-165C, n = 2.5). CTP inhibited both enzymes approximately 70% and ATP activated approximately 40% at the aspartate concentrations required for half-maximal saturation (5 and 90 mM, respectively). The maximal velocity of the mutant holoenzyme is almost identical to that of the wild-type enzyme. The phenylalanine substitution does not affect the stability of the holoenzyme to heat or mercurials, and the Vmax of the catalytic trimer was 444% greater than that of the holoenzyme. Upon dissociation of the wild-type native enzyme into catalytic trimers, the Vmax increased 450%. The Km for aspartate in the separated catalytic trimer is approximately 2-fold higher than for the native catalytic trimer (16.5 versus 8 mM at pH 7.0). It is clear from the data that although Tyr-165C is not directly involved in the active site of the enzyme, it does play a pivotal role in catalytic transitions of the holoenzyme. In addition, the homotropic and heterotropic characteristics of the enzyme do not seem to be altered by the substitution of phenylalanine for Tyr-165C in the E. coli aspartate transcarbamoylase, although other substitutions have been reported (Robey, E. H., and Schachman, H. K. (1984) J. Biol. Chem. 259, 11180-11183) which show more complex effects.
Wales
M E
ME
Department of Biochemistry and Biophysics, Texas A&M University System, College Station 77843.
Hoover
T A
TA
Wild
J R
JR
D001221:Aspartate Carbamoyltransferase / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D006902:Hydroxymercuribenzoates / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D013329:Structure-Activity Relationship; D014443:Tyrosine; D014961:X-Ray Diffraction
D006902:Hydroxymercuribenzoates; C030601:4-hydroxymercuribenzoate; D001224:Aspartic Acid; D014443:Tyrosine; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid