Dynamic Requirements for a Functional Protein Hinge
Identifieur interne : 004A87 ( Main/Exploration ); précédent : 004A86; suivant : 004A88Dynamic Requirements for a Functional Protein Hinge
Auteurs : James G. Kempf [États-Unis] ; Ju-Yeon Jung [États-Unis] ; Christina Ragain [États-Unis] ; Nicole S. Sampson [États-Unis] ; J. Patrick Loria [États-Unis]Source :
- Journal of molecular biology [ 0022-2836 ] ; 2007.
Abstract
The enzyme triosephosphate isomerase (TIM) is a model of catalytic efficiency. The 11-residue loop 6 at the TIM active site plays a major role in this enzymatic prowess. The loop moves between open and closed states, which facilitate substrate access and catalysis, respectively. The N- and C-terminal hinges of loop 6 control this motion. Here, we detail flexibility requirements for hinges in a comparative solution NMR study of wild-type (WT) TIM and a quintuple mutant (PGG/GGG). The latter contained glycine substitutions in the N-terminal hinge at Val167 and Trp168, which follow the essential Pro166, and in the C-terminal hinge at Lys174, Thr175, and Ala176. Previous work demonstrated that PGG/GGG has a 10-fold higher Km value and 103-fold reduced
Url:
DOI: 10.1016/j.jmb.2007.01.074
PubMed: 17336327
PubMed Central: 2203303
Affiliations:
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<front><div type="abstract" xml:lang="en"><title>Summary</title>
<p id="P3">The enzyme triosephosphate isomerase (TIM) is a model of catalytic efficiency. The 11-residue loop 6 at the TIM active site plays a major role in this enzymatic prowess. The loop moves between open and closed states, which facilitate substrate access and catalysis, respectively. The N- and C-terminal hinges of loop 6 control this motion. Here, we detail flexibility requirements for hinges in a comparative solution NMR study of wild-type (WT) TIM and a quintuple mutant (PGG/GGG). The latter contained glycine substitutions in the N-terminal hinge at Val167 and Trp168, which follow the essential Pro166, and in the C-terminal hinge at Lys174, Thr175, and Ala176. Previous work demonstrated that PGG/GGG has a 10-fold higher K<sub>m</sub>
value and 10<sup>3</sup>
-fold reduced <italic>k</italic>
<sub>cat</sub>
relative to WT with either [D]-glyceraldehyde 3-phosphate or dihyrdroxyacetone phosphate as substrate. Our NMR results explain this in terms of altered loop-6 dynamics in PGG/GGG. In the mutant, loop 6 exhibits conformational heterogeneity with corresponding motional rates < 750 s<sup>−1</sup>
that are an order of magnitude slower than the natural WT loop-6 motion. At the same time, ns-timescale motions of loop 6 are greatly enhanced in the mutant relative to WT. These differences from WT behavior occur in both apo PGG/GGG and in the form bound to the reaction-intermediate analog, 2-phosphoglycolate (2-PGA). In addition, as indicated by <sup>1</sup>
H, <sup>15</sup>
N and <sup>13</sup>
CO chemical-shifts, the glycine substitutions (a) diminished the enzyme’s response to ligand, and (b) induced structural perturbations in apo and 2-PGA-bound forms of TIM that are atypical of those observed in WT. Altogether, these data show that PGG/GGG exists in multiple conformations that are not fully competent for ligand binding or catalysis. These experiments elucidate an important principle of catalytic hinge design in proteins: structural rigidity is essential for focused motional freedom of active-site loops.</p>
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