Segmental motion in catalysis: investigation of a hydrogen bond critical for loop closure in the reaction of triosephosphate isomerase

Sampson, Nicole S.; Knowles, Jeremy R.

doi:10.1021/bi00151a015

Cited by 117 publications

(128 citation statements)

References 28 publications

Supporting

Mentioning

119

Contrasting

Order By: Relevance

“…Later experiments suggested that the loop closes after the Michaelis complex is formed, rather than as it is formed (Sampson & Knowles, 1992). This observation is consistent with the paradigm for enzymatic catalysis: that stabilization of intermediates and transition states should be favored over stabilization of substrates and products.…”

supporting

confidence: 66%

Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase

Sun

Sampson

1998

Protein Science

Self Cite

View full text Add to dashboard Cite

We have determined the sequence requirements for a protein hinge in triosephosphate isomerase. The codons encoding the hinge at the C-terminus of the active-site lid of triosephosphate isomerase were replaced with a genetic library of all possible 8,000 amino acid combinations. The most active of these 8,000 mutants were selected using in vivo complementation of a triosephosphate isomerase deficient strain of E. coli, DF502. Approximately 3% of the mutants complement DF502 with an activity that is above 70% of wild-type activity. The sequences of these hinge mutants reveal that the solutions to the hinge flexibility problem are varied. Moreover, these preferences are sequence dependent; that is, certain pairs occur frequently. They fall into six families of similar sequences. In addition to the hinge sequences expected on the basis of phylogenetic analysis, we selected three new families of 3-amino-acid hinges: X(A/S)(L/K/ M), X(aromatic/P-branched)(L/K), and XP(S/N). The absence of these hinge families in the more than 60 known species of triosephosphate isomerase suggests that during evolution, not all of sequence space is sampled, perhaps because there is no neutral mutation pathway to access the other families.

show abstract

supporting

confidence: 66%

Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase

Sun

Sampson

1998

Protein Science

Self Cite

View full text Add to dashboard Cite

show abstract

“…Metabolically, this would have dire consequences by decoupling OAA consumption from PEP synthesis. A similar function has been ascribed to the active site lid domain in TIM, which is thought to sequester the intermediate of that reaction, preventing its hydration, which would lead to the toxic side product methylglyoxal (25,26). Thus, our model for PEPCK catalysis suggests a mechanism by which the closed conformation of the enzyme becomes increasingly thermodynamically favorable as the enzyme progresses along the reaction coordinate.…”

Section: Induced Fit and Conformational Selectionsupporting

confidence: 53%

“…This results in the occlusion of the active site from the bulk solvent (20)(21)(22)(23)(24). These conformational changes, often required to preclude solvent from competing with the catalzyed reaction (25,26), also preclude substrate binding directly to this enzyme form. Therefore, even if we assume that the lowly populated ''active'' form of the conformational selection model is the same as the ''key-lock'' state and that the enzyme samples this state in solution, then substrate cannot bind directly to this state due to steric occlusion of the active site [supporting information (SI) Fig.…”

mentioning

confidence: 99%

Enzymes with lid-gated active sites must operate by an induced fit mechanism instead of conformational selection

Sullivan

Holyoak

2008

Proc. Natl. Acad. Sci. U.S.A.

148

217

View full text Add to dashboard Cite

The induced fit and conformational selection/population shift models are two extreme cases of a continuum aimed at understanding the mechanism by which the final key-lock or active enzyme conformation is achieved upon formation of the correctly ligated enzyme. Structures of complexes representing the Michaelis and enolate intermediate complexes of the reaction catalyzed by phosphoenolpyruvate carboxykinase provide direct structural evidence for the encounter complex that is intrinsic to the induced fit model and not required by the conformational selection model. In addition, the structural data demonstrate that the conformational selection model is not sufficient to explain the correlation between dynamics and catalysis in phosphoenolpyruvate carboxykinase and other enzymes in which the transition between the uninduced and the induced conformations occludes the active site from the solvent. The structural data are consistent with a model in that the energy input from substrate association results in changes in the free energy landscape for the protein, allowing for structural transitions along an induced fit pathway.enzyme dynamics ͉ population shift ͉ phosphoenolpyruvate carboxykinase I t has been 50 years since the original presentation of the induced fit hypothesis by Daniel Koshland (1) that built upon Emil Fisher's key-lock principle (2) and introduced the idea that the inherent dynamic properties of enzymes play an essential role in the processes of molecular recognition and catalysis. Over the last decade, with the advent of new instrumentation and novel experimental approaches there has been a resurgence in investigations focused upon understanding the specific ways in that these dynamic properties influence the ability of enzymes to achieve enormous levels of selectivity and catalytic power (refs. 3-6 and references therein).Presently there are two primary models used to explain the mechanism by which an enzyme can move toward adopting the final key-lock state ( Fig. 1): (i) the induced fit model and (ii) the conformational selection/population shift model (7-14). As defined originally by Koshland (1), the induced fit model states that the achievement of the final key-lock state, that is, the precise orientation of catalytic groups and residues necessary for catalysis, occurs only after changes in protein structure that are induced by the binding of a substrate. This pathway is represented by the equilibrium constants K 1 and K 2 in Fig. 1. Intrinsic to this model is the ability of the enzyme to form an ''encounter complex'' † (Fig. 1B). In this complex the enzyme is liganded identically to the eventual catalytically competent key-lock state; however, the conformational change leading to the active key-lock state has not yet occurred. In contrast, the conformational selection model states that the enzyme exists in multiple conformational states, of which the substrate has a high affinity for the active or key-lock state. In this model the ligand is suggested to bind to this lowly populated high-energy ...

show abstract

“…A different behavior on viscosity has been found for rate-determining events involving diffusion-controlled motions of enzyme portions. As this is an intramolecular event, plots of k cat°/ k cat against /°usually display slopes Ͻ1 because the solvent viscosity effect is damped by an internal friction of protein regions (15). Clearly, the effect of viscosogen on k cat may vary depending on its accessibility and then on its molecular hindrance.…”

Section: A Physical Process Is the Rate-limitingmentioning

confidence: 99%

Structural Flexibility Modulates the Activity of Human Glutathione Transferase P1-1

Ricci

Caccuri

Bello

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

Presteady-state and steady-state kinetic studies performed on human glutathione transferase P1-1 (EC 2.5.1.18) with 1-chloro-2,4-dinitrobenzene as co-substrate indicate that the rate-determining step is a physical event that occurs after binding of the two substrates and before the -complex formation. It may be a structural transition involving the ternary complex. This event can be related to diffusion-controlled motions of protein portions as k cat°/ k cat linearly increases by raising the relative viscosity of the solution. Similar viscosity dependence has been observed for K m GSH , while K m CDNB is independent. No change of the enzyme structure by viscosogen has been found by circular dichroism analysis. Thus, k cat and K m GSH seem to be related to the frequency and extent of enzyme structural motions modulated by viscosity. Interestingly, the reactivity of Cys-47 which can act as a probe for the flexibility of helix 2 is also modulated by viscosity. Its viscosity dependence parallels that observed for k cat and K m GSH , thereby suggesting a possible correlation between k cat , K m GSH, and diffusioncontrolled motion of helix 2. The viscosity effect on the kinetic parameters of C47S and C47S/C101S mutants confirms the involvement of helix 2 motions in the modulation of K m GSH, whereas a similar role on k cat cannot be ascertained unequivocally. The flexibility of helix 2 modulates also the homotropic behavior of GSH in these mutants. Furthermore, fluorescence experiments support a structural motion of about 4 Å occurring between helix 2 and helix 4 when GSH binds to the G-site.Human placental glutathione transferase P1-1 (GST) 1 (EC 2.5.1.18) is a dimeric enzyme composed of two identical subunits each containing one binding site for GSH (G-site) and a second binding site for the electrophilic co-substrate (H-site). Inspection of the three-dimensional structure indicates the presence near the G-site of the irregular ␣ helix 2 (residues 37-46) which is exposed to the solvent (1). Lys-44, a part of this helix, is involved in the binding of GSH. At the end of helix 2 is Cys-47 which is probably linked by ion pair formation with Lys-54. This electrostatic interaction seems important for the correct spatial arrangement of the G-site (2); lack of this bond by replacement of Cys-47 or Lys-54 with Ser or Ala lowers the affinity for GSH and triggers a positive cooperativity toward the binding of GSH (3, 4). We therefore suggested that Cys-47 acts as a hinge which limits the extent or frequency of conformational transitions involving helix 2 (3, 4). In its absence, helix 2 would become more flexible and contact the adjacent subunit via helix 4 thereby inducing the observed cooperativity (4). Several pieces of evidence indicate that the irregular ␣ helix 2 is a flexible region even in the wild-type enzyme; it displays the highest temperature factors among all other regions of domain I (5); moreover, it offers the sole point of attack (Lys-44) for the proteolytic cleavage by trypsin (5). Finally, the strongest evide...

show abstract

Segmental motion in catalysis: investigation of a hydrogen bond critical for loop closure in the reaction of triosephosphate isomerase

Cited by 117 publications

References 28 publications

Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase

Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase

Enzymes with lid-gated active sites must operate by an induced fit mechanism instead of conformational selection

Structural Flexibility Modulates the Activity of Human Glutathione Transferase P1-1

Contact Info

Product

Resources

About