Mutation of lysine 233 to alanine introduces positive cooperativity into tyrosyl-tRNA synthetase.
Tyrosyl-tRNA synthetase from Bacillus stearothermophilus is a dimeric enzyme which displays half-of-sites reactivity with respect to the binding of both tyrosine and ATP. The binding of both substrates follows Michaelis-Menten kinetics. Mutation of lysine 233 to alanine (K233A) decreases the affinity of the active subunit for ATP at both saturating and subsaturating tyrosine concentrations (from the Hill plot, kcat = 0.56 s-1, nH = 1.54, Kd = 372 mM at 50 microM tyrosine). In addition, this mutant displays sigmoidal kinetics (characteristic of positive cooperativity) with respect to the binding of ATP. These two effects can be reversed by the addition of NaCl (0.5 m final concentration) or by a second alanine mutation at either position K230 or T234. The effect of either NaCl or second site mutation is to increase the binding affinity of the K233A mutant for ATP (KATP values are 22 mM for the K233A mutant in the presence of 0.5 M NaCl, 0.16 mM for the K230A/K233A mutant, and 0.14 mM for the K233A/T234A mutant). With the restoration of the tight binding of ATP, Michaelis-Menten kinetics are restored since the kinetic analysis of tyrosyl adenylate formation involves only binding of ATP to the active subunit. It is likely that the physical mechanism for the positive cooperativity present in the K233A mutant actually exists in the wild-type enzyme but is not observed kinetically due to the initial binding of ATP to the active subunit. These results indicate that, in some cases, a decrease in substrate affinity is sufficient to introduce cooperativity into a noncooperative enzyme.
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MRC Unit for Protein Function and Design, University Chemical Laboratory, Cambridge, U.K.
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012965:Sodium Chloride / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014443:Tyrosine / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D014443:Tyrosine; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D014448:Tyrosine-tRNA Ligase; D008239:Lysine; D000409:Alanine