The time scale of the catalytic loop motion in triosephosphate isomerase11Edited by P. E. Wright

Rozovsky, Sharon; McDermott, Ann E.

doi:10.1006/jmbi.2001.4672

Cited by 121 publications

(166 citation statements)

References 37 publications

Supporting

Mentioning

146

Contrasting

Order By: Relevance

“…Biochemical and genetic evidence suggests that the loop, and its precise sequence, is critical for function (42). Kinetic and spectroscopic measurements indicate that the time scale of loop motion matches that of product release and turnover, suggesting that the reaction intermediate is effectively protected from water throughout its lifetime (25,50). How is the loop opening coordinated with the completion of the chemical reaction?…”

Section: Resultsmentioning

confidence: 99%

Optimal alignment for enzymatic proton transfer: Structure of the Michaelis complex of triosephosphate isomerase at 1.2-Å resolution

Jogl

Rozovsky

McDermott

et al. 2002

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

Self Cite

134

281

View full text Add to dashboard Cite

In enzyme catalysis, where exquisitely positioned functionality is the sine qua non, atomic coordinates for a Michaelis complex can provide powerful insights into activation of the substrate. We focus here on the initial proton transfer of the isomerization reaction catalyzed by triosephosphate isomerase and present the crystal structure of its Michaelis complex with the substrate dihydroxyacetone phosphate at near-atomic resolution. The active site is highly compact, with unusually short and bifurcated hydrogen bonds for both catalytic Glu-165 and His-95 residues. The carboxylate oxygen of the catalytic base Glu-165 is positioned in an unprecedented close interaction with the ketone and the ␣-hydroxy carbons of the substrate (C. . . O Ϸ 3.0 Å), which is optimal for the proton transfer involving these centers. The electrophile that polarizes the substrate, His-95, has close contacts to the substrate's O1 and O2 (N. . . O < 3.0 and 2.6 Å, respectively). The substrate is conformationally relaxed in the Michaelis complex: the phosphate group is out of the plane of the ketone group, and the hydroxy and ketone oxygen atoms are not in the cisoid configuration. The ammonium group of the electrophilic Lys-12 is within hydrogen-bonding distance of the substrate's ketone oxygen, the bridging oxygen, and a terminal phosphate's oxygen, suggesting a role for this residue in both catalysis and in controlling the flexibility of active-site loop.T riosephosphate isomerase (TIM) catalyzes the isomerization between dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (GAP). The uphill direction from DHAP to GAP is essential for optimal throughput in the glycolytic pathway. To accomplish this reaction, TIM extracts the pro-R hydrogen from the C1 carbon of DHAP and then stereo-specifically introduces a proton to the C2 carbon ( Fig. 1 a and b; ref. 1). Kinetic isotope effects and isotope ''washout'' experiments suggest that the reaction proceeds through a planar cis-enediol or enediolate intermediate of moderate stability (2, 3), and that the energetic landscape is characterized by several steps of competitive timescales (4). The crucial proton transfers between C1 and C2 atoms of the substrate are most likely carried out by a single base, the side chain carboxylate of Glu-165, whereas His-95 and Lys-12 probably facilitate the transfer of the hydroxyl proton from O1 to O2 (Fig. 1a; ref. 5). Structural studies revealed an ␣͞␤ fold, now known as the TIM-barrel (6-8), and elucidated the geometry of the catalytic residues at the active site and their interactions with the ligands (9).TIM is a textbook case in enzymatic enolization chemistry and has become the subject of landmark spectroscopic and computational studies elucidating the details of the mechanism, the protein motions relevant to chemistry, and the design principles that allow efficient and uphill proton transfer in enzyme active sites. Spectroscopic and mutation studies have focused on the polarization of the substrate by catalytic residues as well as on the...

show abstract

Section: Resultsmentioning

confidence: 99%

Optimal alignment for enzymatic proton transfer: Structure of the Michaelis complex of triosephosphate isomerase at 1.2-Å resolution

Jogl

Rozovsky

McDermott

et al. 2002

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

Self Cite

134

281

View full text Add to dashboard Cite

show abstract

“…That the lid opening rate appears to be commensurate with the k cat for the reverse reaction (12) leaves open the possibility that lid dynamics play a crucial mechanistic role in the function of this enzyme, the rate-limiting step. Indeed, previous NMR experiments have provided evidence correlating the time scales of the relatively small-amplitude intrinsic loop dynamics with the turnover rates in enzymes, including triosephosphate isomerase (7,39), RNase A (9), dihydrofolate (8), and human cyclophilin A (10), hinting that structural dynamics are capable of playing a rate-limiting role in catalysis. However, it is not clear whether an analogous picture can be extended to such large-amplitude conformational fluctuations as are observed in AK.…”

Section: Kinetics Of Lid Movementsmentioning

confidence: 99%

“…Most of our current understanding of structural motions in solution comes from NMR experiments (5) as well as from molecular dynamics simulations (6), approaches that are best suited to study dynamics in the picoto millisecond time scales. Because catalysis in enzymes frequently occurs in the submillisecond to minute time regime, our current understanding of the relationship between enzyme function and conformational dynamics comes from NMR experiments involving relatively localized motions of active site forming loops on the submillisecond time scale (7)(8)(9)(10). However, many enzymes contain active sites located in between domains in which large-amplitude, low-frequency domain motions are required to complete their Michaelis-Menten enzyme-substrate complexes.…”

mentioning

confidence: 99%

Illuminating the mechanistic roles of enzyme conformational dynamics

Hanson

Duderstadt

Watkins

et al. 2007

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

277

430

View full text Add to dashboard Cite

Many enzymes mold their structures to enclose substrates in their active sites such that conformational remodeling may be required during each catalytic cycle. In adenylate kinase (AK), this involves a large-amplitude rearrangement of the enzyme's lid domain. Using our method of high-resolution single-molecule FRET, we directly followed AK's domain movements on its catalytic time scale. To quantitatively measure the enzyme's entire conformational distribution, we have applied maximum entropy-based methods to remove photon-counting noise from single-molecule data. This analysis shows unambiguously that AK is capable of dynamically sampling two distinct states, which correlate well with those observed by x-ray crystallography. Unexpectedly, the equilibrium favors the closed, active-site-forming configurations even in the absence of substrates. Our experiments further showed that interaction with substrates, rather than locking the enzyme into a compact state, restricts the spatial extent of conformational fluctuations and shifts the enzyme's conformational equilibrium toward the closed form by increasing the closing rate of the lid. Integrating these microscopic dynamics into macroscopic kinetics allows us to model lid opening-coupled product release as the enzyme's rate-limiting step.conformational equilibrium ͉ rate-limiting step ͉ single-molecule FRET ͉ adenylate kinase P roteins such as enzymes are flexible with a range of motions spanning from picoseconds for localized vibrations to seconds for concerted global conformational rearrangements (1). Despite their randomly fluctuating environment, in which stochastic collisions with solvent molecules drive changes in tertiary structure, enzymes have evolved to catalyze reactions efficiently and specifically. Indeed, conformational transitions have been postulated to play a central role in enzyme functions in a wide variety of ways, including direct contribution to catalysis (2), allosteric regulation (3), and large-scale conformational changes in response to ligand binding (4). Most of our current understanding of structural motions in solution comes from NMR experiments (5) as well as from molecular dynamics simulations (6), approaches that are best suited to study dynamics in the picoto millisecond time scales. Because catalysis in enzymes frequently occurs in the submillisecond to minute time regime, our current understanding of the relationship between enzyme function and conformational dynamics comes from NMR experiments involving relatively localized motions of active site forming loops on the submillisecond time scale (7-10). However, many enzymes contain active sites located in between domains in which large-amplitude, low-frequency domain motions are required to complete their Michaelis-Menten enzyme-substrate complexes. Even simple questions regarding these transitions remain generally unanswered: What is the number and range of conformational states accessible to enzymes during their catalytic cycle? How does the enzyme's conformation respond to interac...

show abstract

“…To this day, only a small number of enzymes have been shown to rely on essential proximal and/or distal coupled residue motions for catalysis, among which dihydrofolate reductase (5)(6)(7)(8), cyclophilin A (9, 10), liver alcohol dehydrogenase (11)(12)(13)(14), triose-phosphate isomerase (15)(16)(17)(18)(19), and ribonuclease A (20 -22) remain some of the best characterized systems (for recent reviews see Refs. 23 and 24).…”

mentioning

confidence: 99%

NMR Investigation of Tyr105 Mutants in TEM-1 β-Lactamase

Doucet¹,

Savard

Pelletier

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

The existence of coupled residue motions on various time scales in enzymes is now well accepted, and their detailed characterization has become an essential element in understanding the role of dynamics in catalysis. To this day, a handful of enzyme systems has been shown to rely on essential residue motions for catalysis, but the generality of such phenomena remains to be elucidated. Using NMR spectroscopy, we investigated the electronic and dynamic effects of several mutations at position 105 in TEM-1 ␤-lactamase, an enzyme responsible for antibiotic resistance. Even in absence of substrate, our results show that the number and magnitude of short and long range effects on 1 H-15 N chemical shifts are correlated with the catalytic efficiencies of the various Y105X mutants investigated. In addition, 15 N relaxation experiments on mutant Y105D show that several active-site residues of TEM-1 display significantly altered motions on both picosecond-nanosecond and microsecond-millisecond time scales despite many being far away from the site of mutation. The altered motions among various activesite residues in mutant Y105D may account for the observed decrease in catalytic efficiency, therefore suggesting that short and long range residue motions could play an important catalytic role in TEM-1 ␤-lactamase. These results support previous observations suggesting that internal motions play a role in promoting protein function. Enzymes are extremely efficient catalysts that can accelerate biochemical reactions up to a factor of 10 18 when compared with the same uncatalyzed reaction (1). Although considerable progress has been made in understanding enzyme catalysis over the past few years (2, 3), the detailed explanation of this large rate enhancement remains a significant challenge. Historically regarded as relatively static entities, increasing evidence now suggests that enzymes behave as dynamic machines and that motions on various time scales play important roles in promoting enzyme catalysis (4). To this day, only a small number of enzymes have been shown to rely on essential proximal and/or distal coupled residue motions for catalysis, among which dihydrofolate reductase (5-8), cyclophilin A (9, 10), liver alcohol dehydrogenase (11-14), triose-phosphate isomerase (15-19), and ribonuclease A (20 -22) remain some of the best characterized systems (for recent reviews see Refs. 23 and 24). Analogous behavior among other enzymes remains to be elucidated, although the confirmation of such phenomena in structurally and functionally unrelated protein families and folds suggests that this may be a widespread process (24). A general view of such correlation between structure, function, and dynamics in enzymes would greatly improve our current understanding of these powerful catalysts. In this study, we provide experimental evidence supporting the importance of active-site residue motions in the enzyme TEM-1 ␤-lactamase through the characterization of various Y105X mutants by NMR spectroscopy.We have previously investigated th...

show abstract

The time scale of the catalytic loop motion in triosephosphate isomerase11Edited by P. E. Wright

Cited by 121 publications

References 37 publications

Optimal alignment for enzymatic proton transfer: Structure of the Michaelis complex of triosephosphate isomerase at 1.2-Å resolution

Optimal alignment for enzymatic proton transfer: Structure of the Michaelis complex of triosephosphate isomerase at 1.2-Å resolution

Illuminating the mechanistic roles of enzyme conformational dynamics

NMR Investigation of Tyr105 Mutants in TEM-1 β-Lactamase

Contact Info

Product

Resources

About