A structural perspective on the temperature-dependent activity of enzymes

A structural perspective on the temperature-dependent activity of enzymes | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A structural perspective on the temperature-dependent activity of enzymes View ORCID Profile Matthew J. McLeod , View ORCID Profile Sarah A. E. Barwell , View ORCID Profile Todd Holyoak , Robert Edward Thorne doi: https://doi.org/10.1101/2024.08.23.609221 Matthew J. McLeod 1 Cornell University, Ithaca New York , USA . Department of Physics Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Matthew J. McLeod For correspondence: mjm758{at}cornell.edu Sarah A. E. Barwell 2 University of Waterloo , Waterloo Ontario, Canada . Department of Biology Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sarah A. E. Barwell Todd Holyoak 2 University of Waterloo , Waterloo Ontario, Canada . Department of Biology Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Todd Holyoak Robert Edward Thorne 1 Cornell University, Ithaca New York , USA . Department of Physics Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF ABSTRACT Enzymes are biomolecular catalysts whose activity varies with temperature. Unlike for small-molecule catalysts, the structural ensembles of enzymes can vary substantially with temperature, and it is in general unclear how this modulates the temperature dependence of activity. Here multi-temperature X-ray crystallography was used to record structural changes from −20°C to 40°C for a mesophilic enzyme in complex with inhibitors mimicking substrate-, intermediate-, and product-bound states, representative of major complexes underlying the kinetic constant k cat . Both inhibitors, substrates and catalytically relevant loop motifs increasingly populate catalytically competent conformations as temperature increases. These changes occur even in temperature ranges where kinetic measurements show roughly linear Arrhenius/Eyring behavior where parameters characterizing the system are assumed to be temperature independent. Simple analysis shows that linear Arrhenius/Eyring behavior can still be observed when the underlying activation energy / enthalpy values vary with temperature, e.g., due to structural changes, and that the underlying thermodynamic parameters can be far from values derived from Arrhenius/Eyring model fits. Our results indicate a critical role for temperature-dependent atomic-resolution structural data in interpreting temperature-dependent kinetic data from enzymatic systems. One-Sentence Summary Structural data spanning a 60°C temperature range for enzyme complexes mimicking the substrate-, intermediate-, and product-bound states illuminate how small temperature-dependent structural changes may modulate activity and render parameters deduced from Arrhenius/Eyring plots unreliable. INTRODUCTION Chemical reaction rates increase with temperature. For small-molecule catalysts and substrates, increased rates are associated with increased collision velocities and frequencies arising from increased kinetic energy. For enzyme-catalyzed reactions, additional temperature variation in rates may occur due to changes in the conformational ensembles of the enzyme and substrate. Temperature-dependent enzyme reaction rates are typically measured under saturating substrate conditions, where active sites are fully occupied. Turnover ( k cat ) then reports only on steps after formation of the enzyme-substrate (ES) complex and is independent of collision frequency, and effects of increased kinetic energy will only be observed for processes that occur after formation of ES through to product release and regeneration of the free enzyme (E + P). Despite these and other significant mechanistic differences, analysis of k cat as a function of temperature for enzymatic systems is typically based on Arrhenius or Eyring-Polyani (E-P) formalisms, which were derived from small molecule studies and assume the underlying thermodynamic parameters ( Δ H , Δ S ) are temperature-independent. However, enzymatic Arrhenius/Eyring plots can be more complex than their small molecule counterparts. For example, they may be linear at lower temperatures and then curve downward at elevated temperatures, with rates k cat rapidly decreasing above a temperature T opt . This downward curvature is most often attributed to unfolding, and but may be observed even at temperatures well below the unfolding temperature. (for ex. see 1–6) Three models have been proposed to describe this latter behavior: Macromolecular Rate Theory 1 , the Equilibrium Model 7 and a model described by Roy, Schopf and Warshel 6 . Macromolecular Rate Theory (MMRT) suggests that the temperature dependence of enzymatic rates is controlled by the heat capacity change between two states that determine activity. 1 , 8 – 11 Rigidification of the enzyme ensemble as it traverses the conformationally restricted transition state results in a negative heat capacity difference (Δ C p ‡) and downward curvature of the Arrhenius plot after T opt . 1 The Equilibrium Model suggests that downward curvature is due to a shift of the equilibrium defining an enzyme’s conformational ensemble toward increasing population of inactive states as temperature is increased, prior to denaturation. 7 The third model suggests that the more polar GS is more conformationally restricted than the less polar TS. 6 As temperature increases, there is a loosening of the GS that increases its entropy, leading to a decrease in Δ S ‡ , an increase in Δ G ‡ and downward curvature. The first two of these models have been the most discussed, with evidence from kinetic measurements, molecular dynamics simulations, theoretical analysis, and model fitting providing support for each. Testing these models requires observation of functionally relevant changes in conformational ensembles along the reaction coordinate as temperature is changed. More generally, such observations are required to understand observed rate-temperature relationships. A powerful approach to probing temperature-dependent enzyme structure-activity connection is to determine atomic/near atomic resolution X-ray crystallographic structures over a wide temperature range where enzymes exhibit activity. Ideally, the temperature range should span the lower biological limit of roughly −20°C to the denaturation/unfolding temperature, 15 and the crystal form should allow sufficient conformational flexibility to preserve activity. Robust methods for collecting high-quality structural data from “native”, cryoprotectant-free crystals that maintain liquid internal solvent at temperatures down to ∼200 K (−73 °C) have been established. 16 , 17 Methods and hardware for collecting data with minimal crystal dehydration or degradation at up to ∼90°C have also been demonstrated. 18 This ∼160°C data collection temperature range, feasible at high-resolution with X-ray crystallography, is currently difficult to match using any other structural probe. Despite this, only a handful of multi-temperature crystallography studies spanning a mechanistically useful temperature range have been reported. 19 – 26 A recent publication identified only 11 published crystallographic structures at temperatures above 37°C. 27 Here we performed crystallography at −20, 0, 20, and 40°C to probe a mesophilic, GTP-dependent phosphoenolpyruvate carboxykinase from rat cytosol (rcPEPCK) that retains activity in its crystalline form. To observe changes in structure related to k cat , we used complexes representing three states on the reaction coordinate that comprise k cat ( Scheme 1 ). 28 , 29 Download figure Open in new tab Scheme 1: Rat cytosolic PEPCK reaction mechanism and inhibitor complexes. rcPEPCK has been extensively studied both kinetically and structurally, revealing a clear picture of molecular events leading to activity, allowing temperature-dependent changes to be assessed for their impact on activity. 28 – 34 PEPCK is a metabolic enzyme that interconverts oxaloacetic acid (OAA) and phosphoenolpyruvate (PEP) using a metal-cofactor involved in binding and catalysis (M1, typically Mn 2+ - Fig. S1 ), a second nucleotide-associated metal (M2, typically Mg 2+ ), and a phosphoryl donor (either GTP, ATP, or PP i depending on PEPCK class). 35 With respect to the reversible reaction, the data are consistent with a stepwise mechanism where, in the direction of PEP synthesis (OAA→PEP), the reaction is initiated by the decarboxylation of OAA creating an enol-pyruvate intermediate. This intermediate is subsequently phosphorylated by the phosphoryl donor producing PEP ( Scheme 1 ). (reviewed in 35 ) Substrate binding and activity have been shown to be coupled to several dynamic transitions in the enzyme, including a global closure via rotation of the N- and C-terminal domains that reduces the active site cavity’s total volume, as well as loop and residue rearrangements. 32 More specifically it has been shown that the R-(substrate-binding) and P-(nucleotide-binding) loops undergo disorder-to-order transitions upon binding, aiding in orienting the substrate and nucleotide appropriately. 32 The Ω-loop, an active site lid, undergoes a similar essential disorder-order transition as it folds and closes over the active site and is held in place by the adjacent R-loop after the enzyme has undergone substrate-induced global closure and active site remodeling ( Fig. 1 ). 31 , 33 , 36 Lid closure has been shown to be essential to PEPCK function as the lid holds the active site and substrates in a catalytically competent state and protects the enol-pyruvate intermediate from solvent-mediated protonation and the non-productive formation of pyruvate. 36 Download figure Open in new tab Fig. 1. Open and closed conformations of rcPEPCK along the reaction coordinate. Superposition of the open and closed structures (N-terminal domain residues 3-250) of rcPEPCK, indicating conformational changes of A) the global structure and B) the active site. Holo rcPEPCK (open) is shown in steel blue (PDB 2QEW), and its Ω-loop in light grey, R-loop in coral, and P-loop in lilac. Oxalate- (enol-pyruvate intermediate mimic) GTP bound rcPEPCK (closed) is shown in gold (PDB 3DT2) and its Ω-loop in grey, R-loop in firebrick red, and P-loop in purple. Oxalate and GTP are shown bound to the active site and atoms are colored by type (red – oxygen, blue – nitrogen, green – carbon, purple – phosphorous, pink – manganese). Our temperature-dependent structural characterization of rcPEPCK, as well as other previous works indicating structural changes with varying temperature 19 , 20 , 22 , 37 , 38 , prompted us to re-evaluate assumptions made in applying Arrhenius / Eyring-Polanyi (E-P) models to temperature-dependent enzyme kinetic data. Although Arrhenius/E-P plots provide a qualitative descriptor of an enzyme’s free-energy landscape, combined multi-temperature structural and kinetic/biochemical measurements are required for quantitative and mechanistic insight. RESULTS Multi-temperature kinetics An Eyring-Polyani (E-P) plot of k cat for rcPEPCK in the reverse, PEP→OAA, direction ( Fig. 2 , raw data in Data S1 ) showed a decreasing slope with increasing temperatures above ∼25°C ( SI Data S2 ), with a maximum rate at ∼50°C ( T opt ) and a loss of activity at higher temperatures. In the presence of viscogen (glycerol) at constant viscosity (2.4 cp/mPa), k cat decreased at temperatures above ∼50°C but was unaffected at lower temperatures. k cat / K M for the PEP→OAA reaction was determined at four temperatures between 15°C and 55°C ( SI Table S1) but the data are not sufficient to assess the functional variation with temperature. k cat for the OAA→PEP direction could not be reliably determined above 35°C as the non-enzymatic rate of metal-catalyzed decarboxylation of OAA became significant. An Arrhenius plot of the available data (7-37°C) appeared roughly linear (data not presented). Download figure Open in new tab Fig. 2. Eyring-Polanyi plot for rcPEPCK mediated carboxylation of PEP. Reaction rate vs. temperature at saturating concentrations of substrate without (black circle) and with viscogen (grey squares). Linear fit corresponds to 8-25°C temperature range. Error bars (standard error) may be hidden within the data point. Multi-temperature crystallography While all three complexes examined here were inhibited complexes using mimics for the OAA/PEP substrates, structural evidence using authentic substrates with WT and mutant forms of the enzyme suggest that the conformational states sampled in these complexes faithfully reproduce those of the catalytically competent enzyme-substrate complexes ( Fig. 1 and SI Fig. S2 ). 28 , 29 , 34 , 39 In the oxalate(OX)-GTP and phosphoglycolic acid (PGA)-GDP complexes CO 2 is not present. Therefore, the PGA-GDP complex represents a partially occupied encounter/product complex (prior to CO 2 binding / after CO 2 is released) and the OX-GTP complex represents the intermediate state which may or may not have CO 2 present in the authentic enolate-GTP complex but is still conformationally closed. Therefore, both these complexes represent meaningful states along the catalytic trajectory. 28 , 39 rcPEPCK β-sulfopyruvate (βSP)-GTP complex ( Scheme 1 , Complex 1) Both βSP and GTP were fully occupied in the active site at all temperatures. For GTP, the electron density indicated two resolvable conformers between −20°C and 20°C ( Fig. 3 ). One conformer was modeled in a fixed position that has been clearly resolved in the open PEPCK-GTP complex at cryogenic temperatures, and is deemed incompetent for phosphoryl transfer ( IC in Fig. 3 ). 29 The electron density suggests the other conformer changes pose with temperature, and at 40 °C adopts a conformation that has been observed in cryogenic structures of the closed, oxalate-GTP complex, deemed competent for phosphoryl transfer ( C in Fig. 3 ). 28 , 30 At 40°C, there is only evidence for the closed oxalate-GTP complex conformer. The conformation change of GTP occurs by a rotation of both the α-phosphate and ribose out of the plane of the triphosphate moiety. Occupancy refinements, when the B-factors are fixed to the proteins average B-factor, shows the fixed PEPCK-GTP complex conformer is depopulated while the rotating oxalate-GTP like conformer is populated with temperature ( Fig. 3 ). The occupancies for IC:C conformers are 72%:28% at −20°C, 52%:48% at 0°C, 39%:61% at 20°C, and 0%:100% at 40°C. The change in nucleotide conformation was coupled to movement of the P-loop as it shifted towards the M1 metal with increasing temperature - a process previously associated with enzyme/lid closure in cryogenic structures ( SI Fig. S3 ). 28 , 29 , 40 These adjustments lead to various increases and decreases in interatomic distances between GTP and contacting residues ( SI Fig. S4 ). Electron density in the ordered Ω-loop conformation was observed to increase between −20°C and 0°C, remained nearly constant between 0°C and 20°C, and diminished at 40°C (where overall resolution was worse), hinting at possible disordering of the lid at 40°C and above ( Fig 4 , Table S2, and Fig. S5 for βSP-GTP Ω-loop electron density at lower contour). Over the −20°C to 20°C temperature range where data set resolution was nearly constant, the normalized B-factors (B-factor res -B-factor protein ) of residues 94-134 and 228-251, increased with increasing temperature while those of residues 59-93, 255-303, 413-420, 535-557, and 579-590 decreased ( SI Data S3 ). The normalized B-factors for the Ω-loop are consistent with increased order with increasing temperature between −20°C to 20°C evident in the electron density maps. Download figure Open in new tab Fig. 3. Conformational change toward intermediate state with increasing temperature in the βSP-GTP (forward reaction GS) complex. Structures determined at A) −20°C (carbon – ice blue), B) 0°C (light blue), C) 20°C (orange), and D) 40°C (firebrick red) show a shift in GTP conformation as temperature is increased from the fixed, incompetent position to the rotated, competent one (IC and C respectively). The middle panel shows the same structure rotated 90° (looking in-line with nucleotide) to show conformational changes of the α-phosphate. The right panel shows the modeling without electron density with the refined occupancy values for each conformer. Specifically, the α-phosphate and ribose rotate away from the in-line plane of the triphosphate tail. At 40°C, the conformation of the nucleotide is almost identical to E) the 20°C intermediate complex (green – oxalate-GTP). F) Alignment of −20°C and 40°C structures showing the extent of the conformational change. All other atoms are colored by atom-type. Both 2F o −F c (blue – 1σ) and F o −F c (green and red – 3σ) electron density maps are shown. Download figure Open in new tab Fig. 4. Changes in Ω-loop closed conformation occupancy. In each complex, the Ω-loop is modelled in the closed conformation with an occupancy of 1.0. The 2F o −F c (blue) and F o −F c (red/green) maps resulting from refinement are contoured to 1σ and 3σ, respectively. In the βSP-GTP complex, the closed-conformation occupancy of the Ω-loop increases from −20°C to 20°C, and then shows weak evidence of opening/disordering by 40°C ( Fig. S4 ). In the oxalate-GTP complex, the Ω-loop is mostly closed at all temperatures. In the PGA-GDP complex, the closed conformation occupancy of the Ω-loop increases with increasing temperature. rcPEPCK oxalate (OX)-GTP complex (enol-pyruvate-GTP) ( Scheme 1 , Complex 2) In contrast to the temperature-dependent changes noted for the βSP-GTP complex (Complex 1) , a single dominant/resolvable conformer of oxalate and GTP was modeled at all temperatures. The solvent-exposed O2’ of the ribose sugar ( SI Fig. S6 ) and adjacent atoms showed a significant increase in B-factor with increasing temperature, consistent with this region of the nucleotide becoming disordered rather than populating a new conformation. The lack of movement in the triphosphate portion is correlated with a single dominant/resolvable conformation of the P-loop at all temperatures ( SI Fig. S3 ). Strong electron density for the Ω-loop was present at all temperatures ( Fig. 4 ) although a slightly larger B-factor may suggest increased disorder at 40°C ( SI Table S2 ). Consistent with prior cryogenic structures, at all temperatures the OX-GTP complexes had a PEG molecule bound in a cavity between the N- and C-terminal domains of PEPCK; a loop formed by residues 149-154 changed position to accommodate the PEG. In both the βSP-GTP and PGA-GDP complexes, the cavity region was partially disordered but in the dominant/resolved conformation the loop closed off the cavity to prevent PEG binding ( Fig. S7 ). B-factor analysis indicates some minor temperature dependencies in peripheral regions of the enzyme in this complex. ( SI Data S3 ). Residues 95-125 show increased disorder while residues 265-300 show increased order with increased temperature. rcPEPCK phosphoglycolic acid (PGA)-GDP complex (PEP-GDP) ( Scheme 1 , Complex 3) Like the OX-GTP complex, in the PGA-GDP complex at all temperatures, GDP was fully occupied in the active site and single dominant/resolved conformations of the P-loop and nucleotide were observed ( SI Fig. S3 ). The O3’ and other ribose atoms exhibited local temperature-dependent disorder ( SI Fig. S6 ), as in the OX-GTP complex. PGA, on the other hand, exhibited significant conformational change with temperature ( Fig. 5 and Fig. S2 & 8 ). PGA (and PEP) are known to adopt three discrete conformations. 28 , 34 , 39 Two of these exhibit second sphere coordination to the M1 cation and positions the phosphate at too great a distance from the β-phosphate of GDP for phosphoryl transfer to occur, and are thus deemed catalytically incompetent (IC1, IC2) ( Fig. 5 and Fig. S2 & 8 ). 39 In the third conformation, PGA (PEP) directly coordinates to the M1 metal, and the phosphate is positioned in the same location as the γ-phosphate of GTP that is unoccupied when GDP is bound, and is therefore the catalytically competent conformation (C1, Fig. 5 and Fig. S2 & 8 ). 28 , 34 At −20°C, IC1 and IC2 were fully occupied with a distribution of 40%:60%:0% (IC1:IC2:C1), and shifted to a 50%:50%:0% distribution at 0°C. At 20°C, C1 became populated with a 0%:55%:45% distribution, and at 40°C the shift toward the competent state was nearly complete with a 0%:25%:75% distribution. Concomitant with this binding mode change, Y235 shifted from a buried conformation when the incompetent conformations were dominant and rotated toward the active site as the competent conformation was populated ( Fig. 5D ) . The Ω-loop made a similar shift: it largely occupied the open, disordered state at −20°C, and became increasingly ordered and closed as temperature increased ( Fig. 4 ). The only indication of temperature dependency from normalized B-factors was the increase in order of the Ω-loop as temperature increased ( SI Data S3 ). Download figure Open in new tab Fig. 5. Electron density maps for PGA indicate substrate conformational change with temperature. Model and electron density difference maps at A) −20°C, B) 0°C, C) 20°C, D) 40°C. PGA was observed in three distinct conformations: E) two incompetent outer shell states (IC1 and IC2; PGA-GDP P-P interatomic distances of 7.94 and 6.84 Å) and F) one competent state coordinating directly to the M1 metal (C1; P-P interatomic distance 4.84 Å) ( Fig. S1) . As temperature increased, the relative population of the competent conformation increased. Each occupancy was manually refined to minimize the difference density present and the resultant occupancies are labeled (IC1:IC2:C1) for each panel. Y235 is shown in D) indicating the coupled conformational change of the active site with PGA/PEP location. The final 2F o -F c maps (blue) are contoured to 1σ, and F o -F c (red/green) maps are contoured to 3σ. Positive difference density in D) surrounding IC2 was unchanging as its occupancy is increased. DISCUSSION The significance of crystallographic findings depends on constraints imposed by the crystal lattice and how these modify the ensemble of accessible conformational states relative to those of the enzyme in solution. Prior characterization of rcPEPCK crystals has found that the lattice allows multiple conformational states to be observed. For example, all catalytically relevant domain and loop motions including the transition between open and closed states upon soaking with substrates or inhibitor mimics are allowed. The rich history of structure-function investigations of rcPEPCK provides a basis for interpreting temperature-induced changes. 28 – 35 , 39 , 41 – 44 Furthermore, a simplified approach to time-resolved crystallography has recently revealed the hypothesized active conformation of PEP ( vida infra, competent 1 (C1) conformation) generated from OAA and GTP, confirming that rcPEPCK is active in crystallo and that all dynamic modes necessary for activity are accessible ( SI Fig. S2 ). 34 Temperature increases global B-factors but has variable and complex-dependent local effects Nearly all previous “more than one” multi-temperature crystallographic studies have focused on comparison of room temperature ∼298 K (25°C) and cryogenic temperature ∼100 K (−173 °C) structures. These studies have revealed significant changes in the structural ensemble associated with cryocooling and indicate the importance of measuring structures at physiological temperatures in assessing structure-function relationships. 21 , 23 – 25 , 45 – 48 Data collected at temperatures above 100 K and below ∼220 K (−53°C) (near and below the protein-solvent dynamical (or glass) transition, where most functionally salient protein and solvent dynamics are absent) usually add limited information of relevance to understanding mechanism beyond that available at 100 K. 20 Where crystallographic data at several temperatures above the dynamic transition has been collected, only the apo / holo form and one additional complex have been examined, rather than a larger set of complexes representing states along the reaction coordinate. 19 , 20 , 22 , 26 , 49 Multi-temperature cryoEM studies, where grid-containing samples are prepared at different temperatures immediately prior to cryocooling, have examined two systems and revealed domain-scale changes and changes in the ligand pose. 37 , 38 For one system only the apo complex and the ternary “transition state” like complex 37 were examined, at 6 temperatures between 4°C and 70°C and resolutions between 2.1 and 3.0 Å. 37 For the other system, wild-type and mutant versions of a single complex were examined at temperatures of 4, 37 and 42 °C and resolutions of 4.1 - 5.2 Å. Unlike in multi-temperature crystallography where the target temperature is maintained during data collection, multi-temperature cryoEM requires freeze-trapping which perturbs the conformational ensemble. Even though cooling rates of thin-film cryoEM samples are one to two orders of magnitude larger than can be achieved with microcrystals, substantial relaxation of side chains and smaller moieties is still expected, and sample precooling in cold gas present immediately above the liquid ethane introduces uncertainty in the trapped temperature. Previous multi-temperature structural studies revealed two general phenomena which the present data support. First, global average B-factors increase as temperature increases, primarily reflecting increased thermal fluctuations rather than increased static disorder. 20 – 22 , 50 Second, the effects of temperature on the dynamic behavior of local structural elements such as residues and loops depend on the local context of neighboring residues, charge distributions/pKas, solvation, steric interactions, etc. Both increases and decreases in order with increasing temperature are observed. 19 – 21 , 23 Here, the global B-factor for each of three sampled rcPEPCK complexes comprising k cat increased with increasing temperature ( SI Data S4 ). Each complex has a unique temperature dependence, with local disorder either increased or decreased depending on the region examined ( SI Data S3 ). The intermediate OX-GTP complex was the most temperature agnostic, as the global B-factor showed only a small change and the Ω-loop and substrates did not show clear changes in position or occupancy with temperature. In contrast, both the forward (βSP-GTP) and reverse (PGA-GDP) GS complexes showed an increase in Ω-loop (active-site lid) order with increasing temperature, with additional ordering in the PGA-GDP complex but increased disorder in the βSP-GTP complex observed at 40°C ( Fig. 4 , Table S2 and Fig. S3 ). There was also an increase in the normalized B-factor ( Table S2 ) of the Ω-loop of the OX-GTP complex at 40°C, but this does not translate to significantly more disorder ( Fig. 4 ). Coincident with increasing lid order, the P-loop and nucleotide conformations in the βSP-GTP complex change ( Fig. 3 and Fig. S3) . At 40°C, GTP adopts an eclipsed, competent conformation; the γ-phosphate becomes a better leaving group, thus promoting phosphoryl transfer and turnover. 28 , 40 This eclipsed geometry is similar to that observed in the intermediate state (OX-GTP) ( Fig. 3 ). Although this conformational change which aids phosphoryl transfer (second chemical step) occurs in the βSP-GTP complex, before decarboxylation (the first), it will still result in an increase k cat . At high temperatures, phosphoryl transfer can immediately follow decarboxylation because GTP is already in the eclipsed geometry. In contrast, at low temperatures, after decarboxylation GTP must undergo an energetically costly conformational change to the eclipsed state before phosphoryl transfer can occur. This additional conformational change results in an increased time for turnover. In the PGA-GDP complex, PGA shifted from predominantly populating the two incompetent conformations (IC1 and IC2) below 20°C toward increasing occupancy of the competent state at 20 and 40°C — a conformational shift that is required for phosphoryl transfer (C1) ( Fig. 5 and SI Fig. S8 ). Multi-temperature crystallography reveals structural changes influencing kinetic parameters The observed structural changes are directly supported by previous structure-function studies indicating similar changes when rcPEPCK traverses its reaction coordinate. 28 – 31 Together with results from previous cryoEM studies 37 , 38 , the present results sampling enzyme-ligand complexes corresponding to known states comprising k cat suggest that the equilibrium between conformational states shifts so that the population of active states increases with increasing temperature up until some maximum, T opt . This increase in active state population likely contributes to the observed increase in k cat with temperature, as this parameter reflects the energetic barriers of all events on the reaction coordinate following the formation of the enzyme-substrate complex. 51 If one of these events has a significantly slower rate than the others, k cat will be mostly controlled by that event and following events; this is often simplified by assuming that k cat represents this one event. However, in measurements over a large temperature range (e.g., from ∼280 K to 340 K), temperature-dependent structural changes may differentially affect the individual microscopic steps and their relative contributions to k cat , as seen in the different temperature variations of the Ω-loop in each sampled complex. Here, significant differences in the structural data in response to changing temperature of inhibitor complexes representing three meaningful states on the reaction coordinate are observed. These data indicate changes that may plausibly impact rate-determining step(s) at low and high temperatures.Alternative possibilities that could impact rate-determining steps include changes to the dominant reaction mechanism itself, perhaps involving an increase in occupancy of competent conformers with occupancies too low to be discerned in our maps; and changes in the TS structure with temperature. A previous mutational and kinetic study found that rcPEPCK is at least partially rate-limited by phosphoryl transfer (at ambient temperature). 33 The present structural data support this conclusion, as the structural changes observed (eclipsed phosphate geometry of GTP, PGA conformation changing to coordinate with M1, lid closure allowing for chemistry) with increasing temperature all lead to a shift in conformational state that is more favorable for phosphoryl transfer. The intermediate state, represented here by the OX-GTP complex, is resistant to temperature-induced changes. This is not surprising, as the conformational ensemble of this complex must be tightly constrained for the reaction to proceed. Prior work demonstrated that under conditions where the Ω-loop opens prior to phosphorylation of the enolate intermediate, solvent can protonate the intermediate to form a side product, pyruvate. 31 PEPCK likely evolved to ensure robust loop closure throughout the chemical steps essential to maximize the enzyme’s efficacy, consistent with the conformational stability observed for the OX-GTP complex. The induction of the closed conformation in the OX-GTP complex is likely aided by rotation of Y235, as all other active site residues have largely the same positions in the βSP-GTP and OX-GTP complexes ( Fig. S8 ) 28 , by a redistribution of interaction distances at the active site (and beyond), and by the physicochemical properties of the ligand itself. Increased local disorder in the product-complex suggests a new rate-limiting step at high temperatures Arrhenius / E-P plots of k cat ( T ) for rcPEPCK exhibit non-linear behavior at high temperature, with negative curvature leading to a maximum value for k cat ( T ) at a T opt of ∼50°C and slope inversion at temperatures above T opt . As mentioned previously, this non-Arrhenius behavior near T opt is common in enzyme systems, and recent efforts have attempted to create a generalizable model describing it. 1 – 3 , 5 , 6 , 13 , 14 , 52 , 53 Based upon these efforts, negative curvature at temperatures near T opt may be attributed to 1) a change in mechanism, 2) an increase in the barrier that limits rates at high temperatures, or 3) a new barrier along the reaction coordinate becoming dominant. For rcPEPCK, significant downward curvature, starting near ∼40°C, coincides with k cat exhibiting sensitivity to solvent viscosity (∼50°C). A possible interpretation is that a new, non-chemical rate-limiting step has emerged ( Fig. 2 ). Structures at 40 °C provide weak evidence of a shift to product release as the rate limiting step, which would be consistent with a high-temperature viscosity dependence of k cat . Under these assumptions, we speculate that lid ordering prior to chemistry may then become increasingly unfavorable, and this dynamic (and presumably solvent friction-sensitive) step could become rate limiting. Attempts to obtain high-resolution crystallographic data at temperatures above 40°C were not successful due to crystal instability. However, structures at 40°C provide weak evidence that a shift to product release as the rate limiting step may play a role in the high-temperature viscosity dependence of k cat . For the PEP→OAA reaction direction, at 40°C the Ω-loop in the βSP-GTP (product) complex shows hints of increased disorder: a loss of ordered density ( Fig. 4 and Fig. S5 ) in the loop and an increase in B-factor that is larger than expected based solely on the lower resolution of the 40°C data set ( Table S2 ). Disordering of the Ω-loop should aid in product release thus increasing k cat . In the PGA-GDP (substrate) complex, the lid remains closed at 40°C. Given that the Ω-loop has no direct interaction with either the product or substrate complex, it seems plausible that, like the βSP-GTP complex, disorder in the lid may develop above 40°C. Under these assumptions, we speculate that lid ordering prior to chemistry may then become increasingly unfavorable, and this dynamic (and presumably solvent friction-sensitive) step could become rate limiting. Alternatively, new high-temperature conformers may arise which can specifically bind glycerol. This binding may then increase the fraction of inactive conformers. These hypotheses can be tested with molecular dynamics simulations. Linear Arrhenius plots hide a changing free-energy landscape that is revealed through multi-temperature crystallography The temperature dependent activity of related enzymes (e.g., two mutants) is often compared using Arrhenius or Eyring plots. Activation / thermodynamic parameters are extracted from the slope and y -intercept of linear fits. These plots typically report a modest number of data points, each with significant experimental uncertainty, acquired over a limited temperature range. Uncertainty in the slope of such fits arises both from measurement uncertainties and from using a linear fit when the underlying variation may be nonlinear. Uncertainties in the y -intercept are particularly large, as a fit to data spanning, e.g., 0.003 K - 1 <1/T<0.0037 K - 1 must be extrapolated over more than four times that interval to 1/T=0. 54 An implicit assumption in applying the Arrhenius and Eyring models is that, when k cat (or k cat / T varies linearly with temperature over some range, as is the case at low temperature for rcPEPCK, the thermodynamic parameters describing the system are temperature-independent. Thus, a single “apparent” activation entropy Δ H rxn , app and enthalpy Δ H rxn , app (delineated from the true values Δ S rxn and Δ H rxn )) are assumed. This may be a reasonable assumption in small-molecule reaction kinetics. However, hydrophobic interactions, pK a values, and other properties/interactions of relevance to protein structure and dynamics are temperature dependent. As is clear from this and previous multi-temperature structural studies, enzyme structure is temperature dependent. Here we have observed functionally relevant changes in inhibitor-bound active site structures mimicking the complexes sampled by with k cat even in temperature ranges where the corresponding kinetics plots appear to be strictly linear ( SI Data S2 ). Based on these observations for enzyme systems, there is no reason to expect the underlying thermodynamic parameters to remain constant over the temperature ranges typically probed in kinetics experiments. Interpretation of kinetics measurements As will be discussed in more detail elsewhere, a linear E-P fit to the rcPEPCK data (implicitly assuming a temperature-independent free-energy landscape) ( Fig. 2 ) between 8°C (281 K) and 25°C (298 K) gives Δ H rxn , app = 26.3 ± 0.85 kcal/mol and Δ S rxn , app = 0.037 ± 0.003 kcal/mol•°C ( R 2 =0.997). Identical fits to the rcPEPCK data can be obtained by assuming that Δ H rxn at 8°C is 21.30 kcal/mol (19% smaller) and decreases with increasing temperature at a rate of 0.0178 kcal/mol•°C (i.e., by ∼0.25 kcal or ∼1/4 of a hydrogen bond over 14 °C) and a constant Δ S rxn = 0.019 kcal/mol•°C (48% smaller); or by assuming that Δ H rxn at 8°C is 16.3 kcal/mol (39% smaller) and decreases with increasing temperature at a rate of 0.035 kcal/mol•°C (i.e., by ∼0.5 kcal or ∼1/2 of a hydrogen bond over 14 °C) and a constant Δ S rxn = 0.016 kcal/mol•°C (96% smaller). In fact, there is a wide range of physically plausible combinations of linearly varying enthalpies Δ H rxn ( T ) and fixed entropies Δ S rxn (or of simultaneously varying enthalpies and entropies) that can recapitulate the E-P fit to the activity data. In other words, the observed slope and intercept of Arrhenius or E-P plots of k cat ( T ) constrain but do not determine the underlying enthalpies Δ H rxn ( T ) and entropies Δ S rxn ( T ) and their temperature dependencies that determine the observed k cat . This analysis indicates that small temperature variations of underlying thermodynamic / reaction parameters may, in the absence of near-atomic resolution multi-temperature structural information and/or support from other measurements, make quantitative and even qualitative interpretation of parameters derived from Arrhenius / E-P fits to enzyme kinetic data unreliable, with significant consequences for mechanistic interpretations. Qualitative ordering of, e.g., mutant enzymes based on their Arrhenius / Eyring slopes and intercepts may be unreliable. 48 , 55 – 59 Kinetics data only yields Δ G rxn ( T ), a composite of temperature-dependence, and unknown enthalpy and entropy. CONCLUSION Stochastic thermal fluctuations of an enzyme-substrate complex drive the crossing of energetic barriers. Unlike in small molecule catalysis, in enzymes the relevant enthalpy and entropy changes must vary with temperature due to an enzyme’s many degrees of freedom and lability, the many steps along the reaction coordinate 60 contributing to the overall Δ G rxn ( T ), and the temperature variations of physicochemical interactions that govern enzyme structure and activity. Together, these modulate the temperature-dependent activity that would be otherwise be observed if the enzyme were fully “locked down”, with the loss of activity accompanying high temperature unfolding being the most obvious example. Consequently, analysis of kinetic data based on specific mechanistic assumptions (e.g., a temperature-independent free-energy landscape) may be confounded by gradual variations of underlying parameters ( Δ H rxn and Δ S rxn ) with temperature. To better understand the temperature-dependent activity of enzymes, we used multi-temperature crystallography to reveal the structural changes of rat cytosolic PEPCK-inhibitor complexes, an extensively characterized model system. We have identified temperature-dependent changes in the active site and ligand conformations that, based on extensive prior studies, are expected to contribute to the observed increase in k cat with increasing temperature at temperatures Below T opt . Generally, as temperature increases there is a shift from less competent to more competent active site / substrate configurations, contributing to the increase in k cat (and decrease in Δ G rxn ( T )) observed over the same temperature range. These structural changes are observed even within temperature intervals where the corresponding E-P plots appear linear. At the highest temperature probed, weak evidence for increased disorder of the active site lid suggests a shift in this equilibrium back toward chemically incompetent enzyme states, that in turn may be the origin of the negative Arrhenius curvature and loss of activity at higher temperatures. Motivated by these and previous structural observations, it is clear than the assumption of temperature-independent parameters ( E a , A or Δ H rxn , Δ S rxn ) when interpreting Arrhenius or Eyring-Polanyi fits will in general be invalid, even when fitting temperature intervals over which plotted data appears linear. As will be shown elsewhere, small temperature variations of E a and Δ G rxn will have large and correlated effects on the slope and intercept – on and A app / Δ S rxn,app , muddling their mechanistic significance. Effects of these temperature variations may be so large as to render comparisons of fit parameters Δ H rxn , app and Δ S rxn,app obtained from related enzymes, including engineered enzymes and enzymes adapted to different thermal environments, largely meaningless unless observed differences are supported by complementary measurements. Our results point to a key role for atomic resolution multi-temperature structural studies in illuminating fundamental aspects of structure-function relationships, protein design, and thermal adaptation. Funding MJM and the experimental work at Cornell was supported by NIH award 5R01GM127528-04 and by NSF award DBI-2210041. T.H acknowledges support from the Natural Science and Engineering Research Council (NSERC) of Canada. CHEXS is supported by the NSF award DMR-1829070, and the MacCHESS resource is supported by NIGMS award 1-P30-GM124166-01A1 and NYSTAR. Author contributions Conceptualization: MJM, TH, RET Methodology: MJM, SB, RET Investigation: MJM, SB, RET Expression/Purification: MJM, SB Kinetics: MJM Crystallography: MJM Analysis: MJM, RET Visualization: MJM Funding acquisition: TH, RET Project administration: TH, RET Supervision: TH, RET Writing – original draft: MJM, RET Writing – review & editing: MJM, RET, TH Competing interests RET is majority owner and CTO of MiTeGen, which manufactures and sells some of the tools used in this research. Data and materials availability Structural datasets are deposited in the PDB (accession numbers found in Table S3-5 ) and all other data can be made available. Acknowledgments We would like to acknowledge Norman Tran for thoughtful discussions. We would also like to acknowledge CHESS staff, specifically Irina Kriksunov, Bill Miller, and David Schuller. References 1. ↵ Hobbs JK , Jiao W , Easter AD , Parker EJ , Schipper LA , Arcus VL . Change in Heat Capacity for Enzyme Catalysis Determines the Temperature Dependence of Enzyme Catalysed Rates . ACS chemical biology . 2013 ;( 3 ): 2388 – 2393 . doi: 10.1021/cb4005029 OpenUrl CrossRef 2. ↵ Han MH . Non-linear Arrhenius plots in temperature-dependent kinetic studies of enzyme reactions . I. Single transition processes. Journal of Theoretical Biology . 1972 ; 35 : 543 – 568 . doi: 10.1016/0022-5193(72)90150-6 OpenUrl CrossRef PubMed Web of Science 3. ↵ Struvay C , Feller G . Optimization to low temperature activity in psychrophilic enzymes . International Journal of Molecular Sciences . 2012 ; 13 ( 9 ): 11643 – 11665 . doi: 10.3390/ijms130911643 OpenUrl CrossRef PubMed 4. Lee CK , Daniel RM , Shepherd C , et al. Eurythermalism and the temperature dependence of enzyme activity . The FASEB Journal . 2007 ; 21 ( 8 ): 1934 – 1941 . doi: 10.1096/fj.06-7265com OpenUrl CrossRef PubMed Web of Science 5. ↵ Truhlar DG , Kohen A . Convex Arrhenius plots and their interpretation . Proceedings of the National Academy of Sciences of the United States of America . 2001 ; 98 ( 3 ): 848 – 851 . doi: 10.1073/pnas.98.3.848 OpenUrl Abstract / FREE Full Text 6. ↵ Roy S , Schopf P , Warshel A . Origin of the Non-Arrhenius Behavior of the Rates of Enzymatic Reactions . Journal of Physical Chemistry B . 2017 ; 121 ( 27 ): 6520 – 6526 . doi: 10.1021/acs.jpcb.7b03698 OpenUrl CrossRef 7. ↵ Daniel RM , Danson MJ , Eisenthal R . The temperature optima of enzymes: A new perspective on an old phenomenon . Trends in Biochemical Sciences . 2001 ; 26 ( 4 ): 223 – 225 . doi: 10.1016/S0968-0004(01)01803-5 OpenUrl CrossRef PubMed Web of Science 8. ↵ Arcus VL , Mulholland AJ. Temperature, Dynamics, and Enzyme-Catalyzed Reaction Rates . Annu Rev Biophys . 2020 ; 49 : 163 – 180 . doi: 10.1146/annurev-biophys-121219 OpenUrl CrossRef 9. Arcus VL , Prentice EJ , Hobbs JK , et al. On the Temperature Dependence of Enzyme-Catalyzed Rates . Biochemistry . 2016 ; 55 ( 12 ): 1681 – 1688 . doi: 10.1021/acs.biochem.5b01094 OpenUrl CrossRef PubMed 10. Van Der Kamp MW , Prentice EJ , Kraakman KL , Connolly M , Mulholland AJ , Arcus VL . Dynamical origins of heat capacity changes in enzyme-catalysed reactions . Nature Communications . 2018 ; 9 ( 1 ): 1 – 7 . doi: 10.1038/s41467-018-03597-y OpenUrl CrossRef 11. ↵ Walker EJ , Hamill CJ , Crean R , et al. Cooperative Conformational Transitions Underpin the Activation Heat Capacity in the Temperature Dependence of Enzyme Catalysis . ACS Catal . 2024 ; 14 ( 7 ): 4379 – 4394 . doi: 10.1021/acscatal.3c05584 OpenUrl CrossRef 12. Åqvist J , Sočan J , Purg M . Hidden Conformational States and Strange Temperature Optima in Enzyme Catalysis . Biochemistry . 2020 ; 59 ( 40 ): 3844 – 3855 . doi: 10.1021/acs.biochem.0c00705 OpenUrl CrossRef 13. ↵ Åqvist J , Van Der Ent F . Calculation of Heat Capacity Changes in Enzyme Catalysis and Ligand Binding . Journal of Chemical Theory and Computation . 2022 ; 18 ( 10 ): 6345 – 6353 . doi: 10.1021/acs.jctc.2c00646 OpenUrl CrossRef 14. ↵ Daniel RM , Danson MJ . Temperature and the catalytic activity of enzymes: A fresh understanding . FEBS Letters . 2013 ; 587 ( 17 ): 2738 – 2743 . doi: 10.1016/j.febslet.2013.06.027 OpenUrl CrossRef PubMed 15. ↵ Clarke A . The thermal limits to life on Earth . International Journal of Astrobiology . 2014 ; 13 ( 02 ): 141 – 154 . doi: 10.1017/S1473550413000438 OpenUrl CrossRef 16. ↵ Warkentin M , Thorne RE . Glass transition in thaumatin crystals revealed through temperature-dependent radiation-sensitivity measurements . Acta Crystallographica Section D: Biological Crystallography . 2010 ; 66 ( 10 ): 1092 – 1100 . doi: 10.1107/S0907444910035523 OpenUrl CrossRef PubMed 17. ↵ Warkentin M , Thorne RE . Slow cooling of protein crystals . Journal of Applied Crystallography . 2009 ; 42 ( 5 ): 944 – 952 . doi: 10.1107/S0021889809023553 OpenUrl CrossRef PubMed 18. ↵ Doukov T , Herschlag D , Yabukarski F . Instrumentation and experimental procedures for robust collection of X-ray diffraction data from protein crystals across physiological temperatures . Journal of Applied Crystallography . 2020 ; 53 : 1493 – 1501 . doi: 10.1107/S1600576720013503 OpenUrl CrossRef 19. ↵ Keedy DA , Hill ZB , Biel JT , et al. An expanded allosteric network in PTP1B by multitemperature crystallography, fragment screening, and covalent tethering . eLife . 2018 ; 7 : 1 – 36 . doi: 10.7554/eLife.36307.001 OpenUrl CrossRef PubMed 20. ↵ Keedy DA , Kenner LR , Warkentin M , et al. Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography . eLife . 2015 ; 4 : 1 – 26 . doi: 10.7554/eLife.07574 OpenUrl CrossRef PubMed 21. ↵ Fraser JS , Van Den Bedem H , Samelson AJ , et al. Accessing protein conformational ensembles using room-temperature X-ray crystallography . Proceedings of the National Academy of Sciences of the United States of America . 2011 ; 108 ( 39 ): 16247 – 16252 . doi: 10.1073/pnas.1111325108 OpenUrl Abstract / FREE Full Text 22. ↵ Tilton RF , Dewan JC , Petsko GA . Effects of Temperature on Protein Structure and Dynamics: X-ray Crystallographic Studies of the Protein Ribonuclease-A at Nine Different Temperatures from 98 to 320 K . Biochemistry . 1992 ; 31 ( 9 ): 2469 – 2481 . doi: 10.1021/bi00124a006 OpenUrl CrossRef PubMed 23. ↵ Fraser JS , Clarkson MW , Degnan SC , Erion R , Kern D , Alber T . Hidden alternative structures of proline isomerase essential for catalysis . Nature . 2009 ; 462 ( 7273 ):669-673. doi: 10.1038/nature08615 OpenUrl CrossRef PubMed Web of Science 24. Stachowski TR , Vanarotti M , Seetharaman J , Lopez K , Fischer M . Water Networks Repopulate Protein–Ligand Interfaces with Temperature . Angew Chem Int Ed . 2022 ; 61 ( 31 ). doi: 10.1002/anie.202112919 OpenUrl CrossRef 25. ↵ Bradford SYC , El Khoury L , Ge Y , Osato M , Mobley DL , Fischer M . Temperature artifacts in protein structures bias ligand-binding predictions . Chem Sci . 2021 ; 12 ( 34 ): 11275 – 11293 . doi: 10.1039/D1SC02751D OpenUrl CrossRef 26. ↵ Dong M , Lauro ML , Koblish TJ , Bahnson BJ . Conformational sampling and kinetics changes across a non-Arrhenius break point in the enzyme thermolysin . Structural Dynamics . 2020 ; 7 ( 1 ). doi: 10.1063/1.5130582 OpenUrl CrossRef 27. ↵ Guerrero L , Ebrahim A , Riley BT , et al. Pushed to extremes: distinct effects of high temperature versus pressure on the structure of STEP . Commun Biol . 2024 ; 7 ( 1 ): 59 . doi: 10.1038/s42003-023-05609-0 OpenUrl CrossRef 28. ↵ Sullivan SM , Holyoak T . Enzymes with lid-gated active sites must operate by an induced fit mechanism instead of conformational selection . Proceedings of the National Academy of Sciences . 2008 ; 105 ( 37 ): 13829 – 13834 . doi: 10.1073/pnas.0805364105 OpenUrl Abstract / FREE Full Text 29. ↵ Sullivan SM , Holyoak T . Structures of Rat Cytosolic PEPCK: Insight into the Mechanism of Phosphorylation and Decarboxylation of Oxaloacetic Acid . Biochemistry . 2007 ; 46 ( 35 ): 10078 – 10088 . doi: 10.1021/bi701038x OpenUrl CrossRef PubMed 30. ↵ Carlson GM , Holyoak T . Structural insights into the mechanism of phosphoenolpyruvate carboxykinase catalysis . J Biol Chem . 2009 ; 284 ( 40 ): 27037 – 27041 . doi: 10.1074/jbc.R109/040568 OpenUrl FREE Full Text 31. ↵ Johnson TA , Holyoak T . The Ω-loop Lid Domain of Phosphoenolpyruvate Carboxykinase is Essential for Catalytic Function . Biochemistry . 2012 ; 51 ( 47 ): 9547 – 9559 . doi: 10.1021/bi301278t OpenUrl CrossRef PubMed 32. ↵ Holyoak T , Sullivan SM , Nowak T . Structural Insight into the Mechanism of PEPCK Catalysis . Biochemistry . 2006 ; 45 ( 27 ): 8254 – 8263 . doi: 10.1021/bi060269g OpenUrl CrossRef PubMed 33. ↵ Johnson TA , Mcleod MJ , Holyoak T . Utilization of Substrate Intrinsic Binding Energy for Conformational Change and Catalytic Function in Phosphoenolpyruvate Carboxykinase . Biochemistry . 2016 ; 55 ( 3 ): 575 – 587 . doi: 10.1021/acs.biochem.5b01215 OpenUrl CrossRef 34. ↵ Clinger JA , Moreau DW , McLeod MJ , Holyoak T , Thorne RE . Millisecond mix-and-quench crystallography (MMQX) enables time-resolved studies of PEPCK with remote data collection . IUCrJ . 2021 ; 8 ( 5 ): 784 – 792 . doi: 10.1107/s2052252521007053 OpenUrl CrossRef 35. ↵ McLeod MJ , Holyoak T . Phosphoenolpyruvate Carboxykinases . Encyclopedia of Biological Chemistry III . 2021 ; 3 : 400 – 412 . doi: 10.1016/b978-0-12-819460-7.00226-7 OpenUrl CrossRef 36. ↵ Cui DS , Broom A , McLeod MJ , Meiering EM , Holyoak T . Asymmetric Anchoring is Required for Efficient Ω-Loop Opening and Closing in Cytosolic Phosphoenolpyruvate Carboxykinase . Biochemistry . 2017 ; 56 ( 15 ): 2106 – 2115 . doi: 10.1021/acs.biochem.7b00178 OpenUrl CrossRef 37. ↵ Chen CY , Chang YC , Lin BL , Huang CH , Tsai MD . Temperature-Resolved Cryo-EM Uncovers Structural Bases of Temperature-Dependent Enzyme Functions . Journal of the American Chemical Society . 2019 ; 141 ( 51 ): 19983 – 19987 . doi: 10.1021/jacs.9b10687 OpenUrl CrossRef 38. ↵ Singh AK , McGoldrick LL , Demirkhanyan L , Leslie M , Zakharian E , Sobolevsky AI . Structural basis of temperature sensation by the TRP channel TRPV3 . Nature Structural and Molecular Biology . 2019 ; 26 ( 11 ): 994 – 998 . doi: 10.1038/s41594-019-0318-7 OpenUrl CrossRef 39. ↵ Stiffin RM , Sullivan SM , Carlson GM , Holyoak T . Differential Inhibition of Cytosolic PEPCK by Substrate Analogues. Kinetic and Structural Characterization of Inhibitor Recognition . Biochemistry . 2008 ; 47 ( 7 ): 2099 – 2109 . doi: 10.1021/bi7020662 OpenUrl CrossRef PubMed Web of Science 40. ↵ Sudom AM , Prasad L , Goldie H , Delbaere LTJ . The Phosphoryl-transfer Mechanism of Escherichia coli Phosphoenolpyruvate Carboxykinase From the Use of AlF3 . Journal of Molecular Biology . 2001 ; 314 : 83 – 92 . doi: 10.1006/jmbi.2001.5120 OpenUrl CrossRef PubMed Web of Science 41. ↵ Holyoak T , Nowak T . pH Dependence of the Reaction Catalyzed by Avian Mitochondrial Phosphoenolpyruvate Carboxykinase . Biochemistry . 2004 ; 43 ( 22 ): 7054 – 7065 . doi: 10.1021/bi049707e OpenUrl CrossRef PubMed 42. Johnson TA , Holyoak T . Increasing the Conformational Entropy of the Ω-Loop Lid Domain in Phosphoenolpyruvate Carboxykinase Impairs Catalysis and Decreases Catalytic Fidelity . Biochemistry . 2010 ; 49 ( 25 ): 5176 – 5187 . doi: 10.1021/bi100399e OpenUrl CrossRef PubMed 43. Barwell S , Duman R , Wagner A , Holyoak T . Directional regulation of cytosolic PEPCK catalysis is mediated by competitive binding of anions . Biochemical and Biophysical Research Communications . 2022 ; 637 : 218 – 223 . doi: 10.1016/j.bbrc.2022.11.025 OpenUrl CrossRef 44. ↵ Balan MD , McLeod MJ , Lotosky WR , Ghaly M , Holyoak T . Inhibition and Allosteric Regulation of Monomeric Phosphoenolpyruvate Carboxykinase by 3-Mercaptopicolinic Acid . Biochemistry . 2015 ; 54 ( 38 ): 5878 – 5887 . doi: 10.1021/acs.biochem.5b00822 OpenUrl CrossRef 45. ↵ Juers DH , Matthews BW . Reversible lattice repacking illustrates the temperature dependence of macromolecular interactions . Journal of Molecular Biology . 2001 ; 311 ( 4 ): 851 – 862 . doi: 10.1006/jmbi.2001.4891 OpenUrl CrossRef PubMed 46. Skaist Mehlman T , Biel JT , Azeem SM , et al. Room-temperature crystallography reveals altered binding of small-molecule fragments to PTP1B . eLife . 2023 ; 12 : e84632 . doi: 10.7554/eLife.84632 OpenUrl CrossRef 47. Lang PT , Holton JM , Fraser JS , Alber T . Protein structural ensembles are revealed by redefining X-ray electron density noise . Proceedings of the National Academy of Sciences of the United States of America . 2014 ; 111 ( 1 ): 237 – 242 . doi: 10.1073/pnas.1302823110 OpenUrl Abstract / FREE Full Text 48. ↵ Nagel ZD , Dong M , Bahnson BJ , Klinman JP . Impaired protein conformational landscapes as revealed in anomalous Arrhenius prefactors . PNAS . 2011 ; 108 ( 26 ): 10520 – 10525 . doi: 10.1073/pnas.1104989108 OpenUrl Abstract / FREE Full Text 49. ↵ Yabukarski F , Biel JT , Pinney MM , et al. Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray–derived conformational ensembles . Proceedings of the National Academy of Sciences of the United States of America . 2020 ; 117 ( 52 ): 33204 – 33215 . doi: 10.1073/PNAS.2011350117 OpenUrl Abstract / FREE Full Text 50. ↵ Frauenfelder H , Petsko GA . Structural dynamics of liganded myoglobin . Biophysical Journal . 1980 ; 32 : 465 – 483 . doi: 10.1016/S0006-3495(80)84984-8 OpenUrl CrossRef PubMed Web of Science 51. ↵ Northrop DB . On the meaning of Km and V/K in enzyme kinetics . Journal of Chemical Education . 1998 ; 75 ( 9 ): 1153 – 1157 . doi: 10.1021/ed075p1153 OpenUrl CrossRef 52. ↵ Londesborough J . The Causes of Sharply Bent or Discontinuous Arrhenius Plots for Enzyme-Catalysed Reactions . European Journal of Biochemistry . 1980 ; 105 ( 2 ): 211 – 215 . doi: 10.1111/j.1432-1033.1980.tb04491.x OpenUrl CrossRef PubMed Web of Science 53. ↵ Case A , Stein RL . Mechanistic origins of the substrate selectivity of serine proteases . Biochemistry . 2003 ; 42 ( 11 ): 3335 – 3348 . doi: 10.1021/bi020668l OpenUrl CrossRef PubMed 54. ↵ Cornish-Bowden A . Enthalpy-entropy compensation: a phantom phenomenon . J Biosci . 2002 ; 27 ( 2 ): 121 – 126 . doi: 10.1007/BF02703768 OpenUrl CrossRef PubMed 55. ↵ Lenaz G , Sechi AM , Parenti-Castelli G , Landi L , Bertoli E . Activation Energies of Different Mitochondrial Enzymes: Breaks in Arrhenius Plots of Membrane-Bound Enzymes Occur at Different Temperatures . Biochemical and Biophysical Research Communications . 1972 ; 49 ( 2 ): 536 – 542 . doi: 10.1016/0006-291X(72)90444-5 OpenUrl CrossRef PubMed 56. Lonhienne T , Baise E , Feller G , Bouriotis V , Gerday C . Enzyme activity determination on macromolecular substrates by isothermal titration calorimetry: application to mesophilic and psychrophilic chitinases . Biochimica et Biophysica Acta . 2001 ; 1545 : 349 – 456 . doi: 10.1016/S0167-4838(00)00296-X OpenUrl CrossRef PubMed 57. Allen B , Blum M , Cunningham A , Tu GC , Hofmann T . A ligand-induced, temperature-dependent conformational change in penicillopepsin. Evidence from nonlinear Arrhenius plots and from circular dichroism studies . Journal of Biological Chemistry . 1990 ; 265 ( 9 ): 5060 – 5065 . doi: 10.1016/s0021-9258(19)34084-0 OpenUrl Abstract / FREE Full Text 58. Silverstein TP . Falling enzyme activity as temperature rises: Negative activation energy or denaturation? Journal of Chemical Education . 2012 ; 89 ( 9 ): 1097 – 1099 . doi: 10.1021/ed200497r OpenUrl CrossRef 59. ↵ Lam SY , Yeung RCY , Yu TH , Sze KH , Wong KB . A rigidifying salt-bridge favors the activity of thermophilic enzyme at high temperatures at the expense of low-temperature activity . PLoS Biology . 2011 ; 9 ( 3 ). doi: 10.1371/journal.pbio.1001027 OpenUrl CrossRef PubMed 60. ↵ Machado TFG , Gloster TM, da Silva RG. Linear Eyring Plots Conceal a Change in the Rate-Limiting Step in an Enzyme Reaction . Biochemistry . 2018 ; 57 ( 49 ): 6757 – 6761 . doi: 10.1021/acs.biochem.8b01099 OpenUrl CrossRef View the discussion thread. Back to top Previous Next Posted August 23, 2024. Download PDF Supplementary Material Data/Code Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following A structural perspective on the temperature-dependent activity of enzymes Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share A structural perspective on the temperature-dependent activity of enzymes Matthew J. McLeod , Sarah A. E. Barwell , Todd Holyoak , Robert Edward Thorne bioRxiv 2024.08.23.609221; doi: https://doi.org/10.1101/2024.08.23.609221 Share This Article: Copy Citation Tools A structural perspective on the temperature-dependent activity of enzymes Matthew J. McLeod , Sarah A. E. Barwell , Todd Holyoak , Robert Edward Thorne bioRxiv 2024.08.23.609221; doi: https://doi.org/10.1101/2024.08.23.609221 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Biophysics Subject Areas All Articles Animal Behavior and Cognition (7644) Biochemistry (17728) Bioengineering (13916) Bioinformatics (42037) Biophysics (21488) Cancer Biology (18636) Cell Biology (25552) Clinical Trials (138) Developmental Biology (13401) Ecology (19940) Epidemiology (2067) Evolutionary Biology (24367) Genetics (15621) Genomics (22545) Immunology (17764) Microbiology (40475) Molecular Biology (17208) Neuroscience (88744) Paleontology (667) Pathology (2842) Pharmacology and Toxicology (4834) Physiology (7659) Plant Biology (15175) Scientific Communication and Education (2047) Synthetic Biology (4304) Systems Biology (9834) Zoology (2272)