The ‘KMSKS' Motif in Tyrosyl-tRNA Synthetase Participates in the Initial Binding of tRNA^Tyr

Abstract

Variants at each position of the 'KMSKS' signature motif in tyrosyl-tRNA synthetase have been analyzed to test the hypothesis that this motif is involved in catalysis of the second step of the aminoacylation reaction (i.e., the transfer of tyrosine from the enzyme-bound tyrosyl-adenylate intermediate to the tRNA(Tyr) substrate). Pre-steady-state kinetic studies show that while the rate constants for tyrosine transfer (k(4)) are similar to the wild-type value for all of the mobile loop variants, the K230A and K233A variants have increased dissociation constants (K(d)(tRNA)( )()= 2.4 and 1.7 microM, respectively) relative to the wild-type enzyme (K(d)(tRNA)( )()= 0.39 microM). In contrast, the K(d)(tRNA) values for the F231L, G232A, and T234A variants are similar to that of the wild-type enzyme. The K(d)(tRNA) value for a loop deletion variant, Delta(227-234), is similar to that for the K230A/K233A double mutant variant (3.4 and 3.0 microM, respectively). Double mutant free energy cycle analysis indicates there is a synergistic interaction between the side chains of K230 and K233 during the initial binding of tRNA(Tyr) (DeltaDeltaG(int) = -0.74 kcal/mol). These results suggest that while the 'KMSKS' motif is important for the initial binding of tRNA(Tyr) to tyrosyl-tRNA synthetase, it does not play a catalytic role in the second step of the reaction. These studies provide the first kinetic evidence that the 'KMSKS' motif plays a role in the initial binding of tRNA(Tyr) to tyrosyl-tRNA synthetase.

tRNA discrimination by T. thermophilus phenylalanyl–tRNA synthetase at the binding step

“…This result is not unexpected since a characteristic feature of T. thermophilus PheRS is the highest affinity observed for synthetase-tRNA complexes at physiological pH. For a limited number of synthetases the dissociation constants of complexes with cognate tRNAs were found to be in the range 7-14 nM (Glasfeld et al, 1996;Ohannesian and Hou, 1996;Bullock et al, 2000), while most systems have K d values on the order of 0.1-1 mM (for reviews see Meinnel et al, 1995;Gale et al, 1996;Bovee et al, 1999;Ibba et al, 1999;Xin et al, 2000). Furthermore, some of the K d measurements have been performed at low pH, which may increase the binding affinity.…”

tRNA discrimination by T. thermophilus phenylalanyl–tRNA synthetase at the binding step

“…This result is not unexpected since a characteristic feature of T. thermophilus PheRS is the highest affinity observed for synthetase-tRNA complexes at physiological pH. For a limited number of synthetases the dissociation constants of complexes with cognate tRNAs were found to be in the range 7-14 nM (Glasfeld et al, 1996;Ohannesian and Hou, 1996;Bullock et al, 2000), while most systems have K d values on the order of 0.1-1 mM (for reviews see Meinnel et al, 1995;Gale et al, 1996;Bovee et al, 1999;Ibba et al, 1999;Xin et al, 2000). Furthermore, some of the K d measurements have been performed at low pH, which may increase the binding affinity.…”

Backbone Brackets and Arginine Tweezers delineate Class I and Class II aminoacyl tRNA synthetases

“…For Class I TyrRS, mutations of any histidine of the HIGH motif [ 46 ] lead to a decrease in activity, since both residues contribute to the stabilization of the transition state of the reaction [ 79 , 80 ]. The same holds true for Asp-1300 and Gln-1301 which interact with the ribose part of the ligand [ 83 , 85 ].…”

“…The asterisk (*) indicates aaRS Types incorporating noncanonical amino acids. Automatically retrieved [ 77 , 78 ] mutation effects [ 79 – 85 ] are shown as centered shapes. In essence, Class I interactions are mainly hydrogen bonds, while Class II adenosine phosphate-binding is realized by an array of different interaction types.…”

ECOD: identification of distant homology among multidomain and transmembrane domain proteins

“…LeuRS consists of a main enzymatic component (composed of a Rossmann-fold catalytic domain and a class Ia-anticodon binding domain) and four additional flexibly linked domains, one of which is the leucine-specific domain [37]. The leucine-specific (LS) domain is located between the catalytic and anticodon-binding domains, N-terminal to a conserved KMSKS signature motif that binds tRNA [38]. Among known three-dimensional protein structures, the LS domain adopts two distinct topologies.…”

tRNA discrimination by T. thermophilus phenylalanyl–tRNA synthetase at the binding step

“…This result is not unexpected since a characteristic feature of T. thermophilus PheRS is the highest affinity observed for synthetase-tRNA complexes at physiological pH. For a limited number of synthetases the dissociation constants of complexes with cognate tRNAs were found to be in the range 7-14 nM (Glasfeld et al, 1996;Ohannesian and Hou, 1996;Bullock et al, 2000), while most systems have K d values on the order of 0.1-1 mM (for reviews see Meinnel et al, 1995;Gale et al, 1996;Bovee et al, 1999;Ibba et al, 1999;Xin et al, 2000). Furthermore, some of the K d measurements have been performed at low pH, which may increase the binding affinity.…”

Backbone Brackets and Arginine Tweezers delineate Class I and Class II aminoacyl tRNA synthetases

“…For Class I TyrRS, mutations of any histidine of the HIGH motif [ 46 ] lead to a decrease in activity, since both residues contribute to the stabilization of the transition state of the reaction [ 79 , 80 ]. The same holds true for Asp-1300 and Gln-1301 which interact with the ribose part of the ligand [ 83 , 85 ].…”

“…The asterisk (*) indicates aaRS Types incorporating noncanonical amino acids. Automatically retrieved [ 77 , 78 ] mutation effects [ 79 – 85 ] are shown as centered shapes. In essence, Class I interactions are mainly hydrogen bonds, while Class II adenosine phosphate-binding is realized by an array of different interaction types.…”

ECOD: identification of distant homology among multidomain and transmembrane domain proteins

“…LeuRS consists of a main enzymatic component (composed of a Rossmann-fold catalytic domain and a class Ia-anticodon binding domain) and four additional flexibly linked domains, one of which is the leucine-specific domain [37]. The leucine-specific (LS) domain is located between the catalytic and anticodon-binding domains, N-terminal to a conserved KMSKS signature motif that binds tRNA [38]. Among known three-dimensional protein structures, the LS domain adopts two distinct topologies.…”

tRNA discrimination by T. thermophilus phenylalanyl–tRNA synthetase at the binding step

“…This result is not unexpected since a characteristic feature of T. thermophilus PheRS is the highest affinity observed for synthetase-tRNA complexes at physiological pH. For a limited number of synthetases the dissociation constants of complexes with cognate tRNAs were found to be in the range 7-14 nM (Glasfeld et al, 1996;Ohannesian and Hou, 1996;Bullock et al, 2000), while most systems have K d values on the order of 0.1-1 mM (for reviews see Meinnel et al, 1995;Gale et al, 1996;Bovee et al, 1999;Ibba et al, 1999;Xin et al, 2000). Furthermore, some of the K d measurements have been performed at low pH, which may increase the binding affinity.…”

Backbone Brackets and Arginine Tweezers delineate Class I and Class II aminoacyl tRNA synthetases

“…For Class I TyrRS, mutations of any histidine of the HIGH motif [ 46 ] lead to a decrease in activity, since both residues contribute to the stabilization of the transition state of the reaction [ 79 , 80 ]. The same holds true for Asp-1300 and Gln-1301 which interact with the ribose part of the ligand [ 83 , 85 ].…”

“…The asterisk (*) indicates aaRS Types incorporating noncanonical amino acids. Automatically retrieved [ 77 , 78 ] mutation effects [ 79 – 85 ] are shown as centered shapes. In essence, Class I interactions are mainly hydrogen bonds, while Class II adenosine phosphate-binding is realized by an array of different interaction types.…”

ECOD: identification of distant homology among multidomain and transmembrane domain proteins

“…LeuRS consists of a main enzymatic component (composed of a Rossmann-fold catalytic domain and a class Ia-anticodon binding domain) and four additional flexibly linked domains, one of which is the leucine-specific domain [37]. The leucine-specific (LS) domain is located between the catalytic and anticodon-binding domains, N-terminal to a conserved KMSKS signature motif that binds tRNA [38]. Among known three-dimensional protein structures, the LS domain adopts two distinct topologies.…”