Systematic investigation of the link between enzyme catalysis and cold adaptation

Article activity feed

1. 

   Version published to 10.7554/elife.72884 on eLife

   Jan 12, 2022
2. 

   eLife

   Dec 19, 2021

   Author Response:

   Evaluation Summary:

   Are enzymes found in organisms that optimally grow at colder temperatures are more active than the same enzymes found in organisms that optimally grow at warmer temperatures? Here, an assessment of the catalytic constants for approximately 2200 enzymes (obtained from the BRENDA database) showed no correlation between the relative catalytic activity and the optimum growth temperature. Further support for this conclusion was obtained from the measurement of the catalytic constant from a selection of ketosteroid isomerases from organisms that optimally grow between 15 and 46 degrees centigrade. These are interesting results, although the significance with respect to earlier studies has not been clearly explained.

   We have made the relationship between previous work and our work more explicit. Earlier …

   Author Response:

   Evaluation Summary:

   Are enzymes found in organisms that optimally grow at colder temperatures are more active than the same enzymes found in organisms that optimally grow at warmer temperatures? Here, an assessment of the catalytic constants for approximately 2200 enzymes (obtained from the BRENDA database) showed no correlation between the relative catalytic activity and the optimum growth temperature. Further support for this conclusion was obtained from the measurement of the catalytic constant from a selection of ketosteroid isomerases from organisms that optimally grow between 15 and 46 degrees centigrade. These are interesting results, although the significance with respect to earlier studies has not been clearly explained.

   We have made the relationship between previous work and our work more explicit. Earlier studies have used a limited number of specific cases to compare enzyme rates from different organisms (for example, n = 28, Figure 1C, Figure 1D). In this work, we performed a systematic analysis of 2223 enzyme reactions, reducing confirmation bias, and we have clarified this point. Prior work developed physical models about enzyme catalysis but were based on data that do not appear to be representative.

   Reviewer #2 (Public Review):

   The authors are trying to understand how enzymes evolve to best enable organisms to adjust to changes in the temperature of their environment. The paper reports an analysis of 2223 values of kcat from the BRENDA database, for 815 organisms with known optimal growth temperatures, and for which there are at least two variants per reaction. This analysis fails to show the expected preference for values of [(kcat)cold/(kcat)warm] > 1 observed in earlier studies.

   This is a useful attempt to use one large databases to gain insight into how enzymes evolve to enable organisms to adapt to changes in temperature. They have done a good job in curating the BRENDA database to identify data that meets their criteria for analysis.

   There are deficiencies that should be corrected.

   (1) The first concerns the reported values of [(kcat)cold/kcat)warm]. Figure 1D shows "Rate comparisons of warm-adapted and cold-adapted enzyme variants made at identical temperatures." I think that it is important that these kinetic parameters be reported for catalysis at a common temperature, but it is not clear to me that is the case for the author's analysis. For example, they write beginning on line 234 that "The rate ratio kcold/kwarm per reaction was determined by dividing rate of the enzyme from the organism with the minimum TGrowth by the rate of the enzyme from organism with the maximum TGrowth." My reading of this sentence is that these rate constants kcat [not rates] were determined individually at the organisms optimal growth temperatures, and not at identical temperatures as reported in Figure 1D. This will complicate the author's interpretation of the two sets of results.

   Analysis of kinetic parameters at a common temperature supports the conclusions of this work.

   (2) The author's fail to present a clear physical model to use in analyzing these results.

   For example, they write on line 35 that: "According to the rate compensation model of temperature adaptation, this challenge is met by cold-adapted enzyme variants providing more rate enhancement than the corresponding warm-adapted variants (Figure 1A)"

   I cannot recall hearing the term rate compensation model, but am familiar with discussions on the differences in properties of enzymes isolated from organisms that have adapted to warm and cold environments. The term cold adapted enzymes is not appropriate, because it is the organism not the enzyme, that adapts to the change to a cold environment. This is accomplished through the natural selection of enzymes with kinetic parameters, stability, etc. that optimize the organisms chances of survival in a cold climate. The kinetic parameters for essentially all enzymes will decrease with decreasing temperature. The most highly evolved metabolic enzymes have kinetic parameters kcat/Km close to the diffusion controlled limit, because this optimizes energy production from metabolism. A decrease in temperature will cause the values of kcat and therefore kcat/Km for these enzymes to decrease, to the detriment of the organism. This may be overcome by selection of enzymes with values of kcat/Km close to that observed for the parent [unevolved] organism. The result is that larger kinetic parameters kcat, for catalysis at a common temperature, will be observed for enzymes isolated from the cold-adapted, compared to the unevolved parent organism. This simple application of Darwin's principals of natural selection is strongly supported by the data reported in Figure 1D.

   The reviewer presents a model that presumes that there would be greater selection to optimize energy production. This is also the model supported by the prior data (Figure 1D).

   However, the more extensive data in our work do not support the model that the reviewer notes and that has been widely accepted in the literature –this is the central conclusion of this work and we have attempted to clarify this, as noted above. The strict Darwinian interpretation for our observations is that there is not a strong selection for enzyme rates to be maximized, as described in the Discussion.

   An alternative model, consistent with the data we present, is that there are different selective pressures on enzymes than rate maximization. We note that it is possible that different metabolic strategies may be more advantageous at different life stages or in different communities (see Wortel et al., 2018, now cited in our main text). These models can be tested experimentally –e.g., by examining how variations of a weak-link enzyme fare over time under different growth conditions. There is much more to be learned from linking the properties of enzymes to evolution, and we expect the relationship between fundamental rate constants and selection to be complex, fascinating and important.

   We use the term rate compensation to refer to the phenomenon and not the physical explanation; there is no need for a physical explanation of a phenomenon in the absence of evidence for the phenomenon itself. We have clarified that we have introduced this term in the Introduction: According to what we term the rate compensation model of temperature adaptation, this challenge has been suggested to be met by cold-adapted enzyme variants providing more rate enhancement than the corresponding warm-adapted variants (Figure 1A).

   We use the term “cold-adapted” in agreement with literature usage: from an organism that is cold adapted. We have clarified this language usage: We use the term “cold-adapted variant” to refer to an enzyme from an organism annotated with lower TGrowth values.

   Finally, “cold-adapted” is not synonymous with “having faster enzymes”, which is often how it is used in the literature and how it is implied in the reviewer’s model.

   (3) The paper alludes to, but does not clearly explain extensions of these ideas that are based on one model for how enzymes work. Enzymes often undergo large conformational changes during their catalytic cycle, and so must have sufficient flexibility for these changes to occur with rate constants that support catalysis. This predicts that the enhancement for catalysis observed for enzymes from cold-adapted organisms, might best be achieved through mutations that favor an increase in protein flexibility. There will also be natural selection of enzymes for thermophilic organisms that optimize the organisms chances of survival in a hot climate, where heat denaturation of the protein catalyst is minimized through the selection of stiffer protein catalysts. This analysis predicts a decrease in enzyme flexibility with increasing preferred growth temperature, that might give rise to an increase in protein stability with increasing optimal growth temperature.

   We agree that there are many fascinating aspects of temperature adaptation at the level of individual enzymes, their mechanisms, and their particular rate-limiting steps that remain to be explored. These were not the subject of our study. The goal of our manuscript was to test the previously presented rate compensation model of enzyme cold adaptation.

   (4) The authors should consider the possibility that the pressure to compensate for the cold-induced decrease in kcat for enzymes from cold-adapted organism will be strongest for highly evolved metabolic enzymes with values of kcat/Km close to the diffusion controlled limit. In cases where the enzyme starts out as less than perfect, an organism adapting to the cold might derive smaller, or even negligible advantages, from natural-selection of enzymes with enhanced kinetic parameters. For example, the organism might also minimize the effect of this change in kinetic parameter, by an adjustment or diversion of flux through the networks of metabolic pathways in which the enzyme functions. One possible explanation for the weak correlation observed between kcat and Tgrowth for ketosteroid isomerase is that the organisms studied gain little from optimization of the activity of this enzyme in cold-adapted organisms. One risk in the use of the larger BRENDA database may be the failure to account for differences in the pressure for enzymes to evolve to enable organisms adapt to cold environments.

   We considered these and additional models. For example, interestingly, the opposite of what the reviewer proposed has been suggested in the literature –that the slowest enzymes (“least perfect”) are under the heaviest selection pressure for optimization (see Noda-Garcia et al., 2018). Although our data indicates that temperature exerts a weaker force on enzyme activity than previously proposed, it is indeed possible that subgroups of enzymes do indeed adapt to temperature through changes in activity. Deciphering this and other pressures is an important future challenge. We did not parse the data in this report out of concerns for “p-hacking” or multiple hypothesis testing.

   Reviewer #3 (Public Review):

   Enzyme catalysis underlies all living processes. Understanding the effects of temperature on enzymes is important in understanding how they are adapted to particular environmental conditions, and also relates to the response of organisms and even ecosystems to changes in temperature. The essential question is: what determines optimal growth rates of organisms, and the optimal temperature of other biological processes? Two potentially important factors are enzyme stability and catalytic activity.

   This manuscript collates data from previous investigations and presents new results on KSI variants, aiming to look at the interesting question of what factors are important in relating enzyme activity and stability to optimum growth temperatures of organisms. It presents a useful survey of published data, particularly focusing on the enzyme ketosteroid isomerase (KSI) for which new resluts for a number of variants are presented, building on nice recent work by this group. The main finding in this manuscript is that enzyme optimum temperatures do not correlate well with enzyme activity. This has been found also previously. The manuscript provides quite an extensive analysis and is consistent with previous results and findings. There is useful information in this manuscript, and the compilation of data will be useful to the community, but some crucial aspects and recent relevant work are not covered, and the discussion is limited. The analysis does not identify any relevant determinant of optimum temperature, and the focus on a single temperature in each case may be misleading.

   We do not agree that our analysis is “misleading.” We would characterize the prior analysis based on a small number of examples that were not randomly selected as potentially misleading. In contrast, we tested the prior conclusions with all relevant data that are available. We also highlight the power of collecting more data by further reporting the rate enhancement of 20 enzyme variants in depth. Temperature compensation through activity may still occur in specific settings, as we have noted in the Discussion.

   We agree with Reviewer #3 about the vast potential to use temperature dependencies to relate to evolutionary pressures and adaptations from molecules to organisms. This is a prime area for future investigation.

   Previous analyses have shown that optimum rates of enzymes do not correlate with optimal growth temperatures (e.g. Elias et al (2014) Trends in Biochemical Sciences 39, 299; Peterson (2004) Journal of Biological Chemistry 279, 20717; Thomas & Scopes (1998) Biochemical Journal 330, 1087; Lee et al (2007) FASEB Journal 21, 1934). This is particularly notable for psychrophilic (cold adapted) enzymes, but is also apparent from the fact that enzymes from the same organism often have quite different optimum temperatures. The data collected in the current manuscript are consistent with previous analyses and so are usefully confirming of this. The authors note that optimal growth temperatures may not correlate with activity for a number of reasons, including that the individual enzyme rate may not be under evolutionary pressure. Also, obviously, as noted by the authors, factors other than temperature are also important in enzyme evolution.

   We agree that it is obvious that factors other than temperature are important in evolution, but here we address whether the adaptation to temperature is accompanied by a common response. As noted, more catalysis for organisms at lower temperature was concluded previously and (as noted by Reviewer #2) is expected. However, this conclusion, upon further analysis (carried out herein) appears not to hold. Thus, even when organisms are adapting to temperature, other factors appear to be dominant. This was not previously known. The analyses the reviewer notes refer to thermal parameters derived from the temperature dependence of the rate constant for a given enzyme as a function of temperature, rather than what is addressed herein –the relative rate constant for enzymes from organisms with different growth temperatures.

   There is somewhat better correlation of enzyme stability with optimum growth temperatures, but it is not strong. Therefore, other factors must be important in determining optimum growth temperatures. The authors briefly mention some possibilities. One factor is that a given enzyme may not be a bottleneck in a metabolic pathway. It is not clear that KSI is in fact a metabolic limiter. Also, for many metabolic pathways, it may be essential to consider the kinetics of the pathway as a whole, which may not be determined by a single enzyme. Directly relevant here is the recent proposal of the 'inflection point hypothesis', which provides an explanation of these observations (Prentice et al. Biochemistry (2020) 59, 3562), which the authors do not mention, and may not be aware of. This hypothesis proposes that, rather than alignment of optimum temperatures or stabilities, rather the inflection points of enzymes in a metabolic pathways are aligned at the mean environmental temperature for the organism. This has the effect of coordinating relative enzyme rates and preventing metabolic disruption as temperature fluctuates. Also relevant here is that the response of metabolic pathways in general is not determined solely by a single enzyme. Prentice et al. show that, in general, the temperature-dependent properties of each enzyme in the pathway is important in determining the temperature dependence of the whole pathway.

   We thank Reviewer #3 for bringing this work to our attention and we have included it in the revised manuscript. This paper points out additional complexities regarding metabolic coordination of relative enzyme rates, enhancing points made in the Discussion.

   It is certainly important to understand what molecular features determine the temperature dependence of enzyme activity and its relationship to stability. Some previous proposals are mentioned in the manuscript. One important factor at the molecular level, mentioned by the authors, is work of Åqvist, Brandsval and coworkers, who have convincingly shown that activation entropy and enthalpy differ significantly between psychrophilic enzymes and their mesophilic and thermophilic counterparts. For small soluble enzymes, this is particularly due to changes at the enzyme surface, which may also affect stability. As mentioned by the authors, there have been many proposals over the years that suggest a relationship between stability and activity, though there is not a simple general relationship.

   The cited study is based on molecular dynamics simulations and underlying potentials which can provide models to be tested via experiment. Our analyses relate to this model in that they suggest that rate compensation (to temperature) is not general and so a universal linkage of temperature, flexibility and catalysis is not expected.

   Also directly relevant for the discussion here is what factors limit enzyme activity as temperature increases. The traditional view is that loss of activity is due to protein unfolding at high temperatures (the poor correlation of stability with growth temperatures found here indicates that this cannot be a general explanation). There is increasing evidence that this simple picture is wrong (see e.g. Daniel & Danson. (2010) Trends in Biochemical Sciences 35, 584). This behavior may be accounted for by conformational (e.g. two state) effects as proposed by Danson et al, distinct from the 'flexibility' proposals mentioned in the supporting information here. The introduction of the manuscript here states that "reaction rates are reduced at lower temperatures" , which might naively seem obvious but actually is not universally true, many reactions do not display simple Arrhenius-type behavior (see e.g. Kohen and Truhlar PNAS 2001 98 848). Many enzymes show a temperature of optimum activity, i.e. activity drops above the optimum temperature but before unfolding occurs. As the authors note, Arcus et al. show that this can be accounted for by an activation heat capacity, significantly larger in psychrophiles. Signatures of this behavior are apparent at the large scale (e.g. Schipper et al Global Change Biol. 2014 20 3578; Alster et al (2016) Front. Microbiol. 7:1821) and it appears to be generally important.

   We also are enthralled by the many proposals put forward for the physical and thermodynamic behavior of enzymes and we look forward to rigorous tests of the predictions of these models. Like Reviewer #3, we expect that there are many different features and properties of enzymes to discover!

   Read the original source
3. 

   eLife

   Dec 19, 2021

   Evaluation Summary:

   Are enzymes found in organisms that optimally grow at colder temperatures are more active than the same enzymes found in organisms that optimally grow at warmer temperatures? Here, an assessment of the catalytic constants for approximately 2200 enzymes (obtained from the BRENDA database) showed no correlation between the relative catalytic activity and the optimum growth temperature. Further support for this conclusion was obtained from the measurement of the catalytic constant from a selection of ketosteroid isomerases from organisms that optimally grow between 15 and 46 degrees centigrade. These are interesting results, although the significance with respect to earlier studies has not been clearly explained.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the …

   Evaluation Summary:

   Are enzymes found in organisms that optimally grow at colder temperatures are more active than the same enzymes found in organisms that optimally grow at warmer temperatures? Here, an assessment of the catalytic constants for approximately 2200 enzymes (obtained from the BRENDA database) showed no correlation between the relative catalytic activity and the optimum growth temperature. Further support for this conclusion was obtained from the measurement of the catalytic constant from a selection of ketosteroid isomerases from organisms that optimally grow between 15 and 46 degrees centigrade. These are interesting results, although the significance with respect to earlier studies has not been clearly explained.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

   Read the original source
4. 

   eLife

   Dec 19, 2021

   Reviewer #1 (Public Review):

   Are enzymes found in organisms that optimally grow at lower temperatures more active than the same enzymes found in organisms that grow at higher temperatures? Herschlag and colleagues have obtained the catalytic constants for a large number of enzymes from public databases and compared the relative magnitude of these constants with the optimal growth temperature. For a group of approximately 2200 enzymes they found no correlation between the ratio of activities for the enzymes from the cold adapted organisms relative to those from organisms that optimally grow at the higher temperatures (Figure 2C). The distribution exhibits an approximately equal number of enzymes that were more active from the higher temperature organisms and those that were less active from the higher temperature organisms, relative to …

   Reviewer #1 (Public Review):

   Are enzymes found in organisms that optimally grow at lower temperatures more active than the same enzymes found in organisms that grow at higher temperatures? Herschlag and colleagues have obtained the catalytic constants for a large number of enzymes from public databases and compared the relative magnitude of these constants with the optimal growth temperature. For a group of approximately 2200 enzymes they found no correlation between the ratio of activities for the enzymes from the cold adapted organisms relative to those from organisms that optimally grow at the higher temperatures (Figure 2C). The distribution exhibits an approximately equal number of enzymes that were more active from the higher temperature organisms and those that were less active from the higher temperature organisms, relative to the lower temperature organisms. Further support for their conclusion was obtained from the measurement of the catalytic constants from a selection of ketosteroid isomerases (KSI) from organisms that optimally grow between 15 and 46{degree sign}C. No correlation was apparent (Figure 3D). Overall, this is a nice contribution that directly addresses whether or not there appears to be any support for the notion that enzymes from cold adapted organisms are more catalytically active than those enzymes from organisms that grow at higher temperatures.

   Read the original source
5. 

   eLife

   Dec 19, 2021

   Reviewer #2 (Public Review):

   The authors are trying to understand how enzymes evolve to best enable organisms to adjust to changes in the temperature of their environment. The paper reports an analysis of 2223 values of kcat from the BRENDA database, for 815 organisms with known optimal growth temperatures, and for which there are at least two variants per reaction. This analysis fails to show the expected preference for values of [(kcat)cold/(kcat)warm] > 1 observed in earlier studies.

   This is a useful attempt to use one large databases to gain insight into how enzymes evolve to enable organisms to adapt to changes in temperature. They have done a good job in curating the BRENDA database to identify data that meets their criteria for analysis.

   There are deficiencies that should be corrected.

   (1) The first concerns the reported values …

   Reviewer #2 (Public Review):

   The authors are trying to understand how enzymes evolve to best enable organisms to adjust to changes in the temperature of their environment. The paper reports an analysis of 2223 values of kcat from the BRENDA database, for 815 organisms with known optimal growth temperatures, and for which there are at least two variants per reaction. This analysis fails to show the expected preference for values of [(kcat)cold/(kcat)warm] > 1 observed in earlier studies.

   This is a useful attempt to use one large databases to gain insight into how enzymes evolve to enable organisms to adapt to changes in temperature. They have done a good job in curating the BRENDA database to identify data that meets their criteria for analysis.

   There are deficiencies that should be corrected.

   (1) The first concerns the reported values of [(kcat)cold/kcat)warm]. Figure 1D shows "Rate comparisons of warm-adapted and cold-adapted enzyme variants made at identical temperatures." I think that it is important that these kinetic parameters be reported for catalysis at a common temperature, but it is not clear to me that is the case for the author's analysis. For example, they write beginning on line 234 that "The rate ratio kcold/kwarm per reaction was determined by dividing rate of the enzyme from the organism with the minimum TGrowth by the rate of the enzyme from organism with the maximum TGrowth." My reading of this sentence is that these rate constants kcat [not rates] were determined individually at the organisms optimal growth temperatures, and not at identical temperatures as reported in Figure 1D. This will complicate the author's interpretation of the two sets of results.

   (2) The author's fail to present a clear physical model to use in analyzing these results.

   For example, they write on line 35 that: "According to the rate compensation model of temperature adaptation, this challenge is met by cold-adapted enzyme variants providing more rate enhancement than the corresponding warm-adapted variants (Figure 1A)"

   I cannot recall hearing the term rate compensation model, but am familiar with discussions on the differences in properties of enzymes isolated from organisms that have adapted to warm and cold environments. The term cold adapted enzymes is not appropriate, because it is the organism not the enzyme, that adapts to the change to a cold environment. This is accomplished through the natural selection of enzymes with kinetic parameters, stability, etc. that optimize the organisms chances of survival in a cold climate. The kinetic parameters for essentially all enzymes will decrease with decreasing temperature. The most highly evolved metabolic enzymes have kinetic parameters kcat/Km close to the diffusion controlled limit, because this optimizes energy production from metabolism. A decrease in temperature will cause the values of kcat and therefore kcat/Km for these enzymes to decrease, to the detriment of the organism. This may be overcome by selection of enzymes with values of kcat/Km close to that observed for the parent [unevolved] organism. The result is that larger kinetic parameters kcat, for catalysis at a common temperature, will be observed for enzymes isolated from the cold-adapted, compared to the unevolved parent organism. This simple application of Darwin's principals of natural selection is strongly supported by the data reported in Figure 1D.

   (3) The paper alludes to, but does not clearly explain extensions of these ideas that are based on one model for how enzymes work. Enzymes often undergo large conformational changes during their catalytic cycle, and so must have sufficient flexibility for these changes to occur with rate constants that support catalysis. This predicts that the enhancement for catalysis observed for enzymes from cold-adapted organisms, might best be achieved through mutations that favor an increase in protein flexibility. There will also be natural selection of enzymes for thermophilic organisms that optimize the organisms chances of survival in a hot climate, where heat denaturation of the protein catalyst is minimized through the selection of stiffer protein catalysts. This analysis predicts a decrease in enzyme flexibility with increasing preferred growth temperature, that might give rise to an increase in protein stability with increasing optimal growth temperature.

   (4) The authors should consider the possibility that the pressure to compensate for the cold-induced decrease in kcat for enzymes from cold-adapted organism will be strongest for highly evolved metabolic enzymes with values of kcat/Km close to the diffusion controlled limit. In cases where the enzyme starts out as less than perfect, an organism adapting to the cold might derive smaller, or even negligible advantages, from natural-selection of enzymes with enhanced kinetic parameters. For example, the organism might also minimize the effect of this change in kinetic parameter, by an adjustment or diversion of flux through the networks of metabolic pathways in which the enzyme functions. One possible explanation for the weak correlation observed between kcat and Tgrowth for ketosteroid isomerase is that the organisms studied gain little from optimization of the activity of this enzyme in cold-adapted organisms. One risk in the use of the larger BRENDA database may be the failure to account for differences in the pressure for enzymes to evolve to enable organisms adapt to cold environments.

   Read the original source
6. 

   eLife

   Dec 19, 2021

   Reviewer #3 (Public Review):

   Enzyme catalysis underlies all living processes. Understanding the effects of temperature on enzymes is important in understanding how they are adapted to particular environmental conditions, and also relates to the response of organisms and even ecosystems to changes in temperature. The essential question is: what determines optimal growth rates of organisms, and the optimal temperature of other biological processes? Two potentially important factors are enzyme stability and catalytic activity.

   This manuscript collates data from previous investigations and presents new results on KSI variants, aiming to look at the interesting question of what factors are important in relating enzyme activity and stability to optimum growth temperatures of organisms. It presents a useful survey of published data, …

   Reviewer #3 (Public Review):

   Enzyme catalysis underlies all living processes. Understanding the effects of temperature on enzymes is important in understanding how they are adapted to particular environmental conditions, and also relates to the response of organisms and even ecosystems to changes in temperature. The essential question is: what determines optimal growth rates of organisms, and the optimal temperature of other biological processes? Two potentially important factors are enzyme stability and catalytic activity.

   This manuscript collates data from previous investigations and presents new results on KSI variants, aiming to look at the interesting question of what factors are important in relating enzyme activity and stability to optimum growth temperatures of organisms. It presents a useful survey of published data, particularly focusing on the enzyme ketosteroid isomerase (KSI) for which new resluts for a number of variants are presented, building on nice recent work by this group. The main finding in this manuscript is that enzyme optimum temperatures do not correlate well with enzyme activity. This has been found also previously. The manuscript provides quite an extensive analysis and is consistent with previous results and findings. There is useful information in this manuscript, and the compilation of data will be useful to the community, but some crucial aspects and recent relevant work are not covered, and the discussion is limited. The analysis does not identify any relevant determinant of optimum temperature, and the focus on a single temperature in each case may be misleading. Previous analyses have shown that optimum rates of enzymes do not correlate with optimal growth temperatures (e.g. Elias et al (2014) Trends in Biochemical Sciences 39, 299; Peterson (2004) Journal of Biological Chemistry 279, 20717; Thomas & Scopes (1998) Biochemical Journal 330, 1087; Lee et al (2007) FASEB Journal 21, 1934). This is particularly notable for psychrophilic (cold adapted) enzymes, but is also apparent from the fact that enzymes from the same organism often have quite different optimum temperatures. The data collected in the current manuscript are consistent with previous analyses and so are usefully confirming of this. The authors note that optimal growth temperatures may not correlate with activity for a number of reasons, including that the individual enzyme rate may not be under evolutionary pressure. Also, obviously, as noted by the authors, factors other than temperature are also important in enzyme evolution.

   There is somewhat better correlation of enzyme stability with optimum growth temperatures, but it is not strong. Therefore, other factors must be important in determining optimum growth temperatures. The authors briefly mention some possibilities. One factor is that a given enzyme may not be a bottleneck in a metabolic pathway. It is not clear that KSI is in fact a metabolic limiter. Also, for many metabolic pathways, it may be essential to consider the kinetics of the pathway as a whole, which may not be determined by a single enzyme. Directly relevant here is the recent proposal of the 'inflection point hypothesis', which provides an explanation of these observations (Prentice et al. Biochemistry (2020) 59, 3562), which the authors do not mention, and may not be aware of. This hypothesis proposes that, rather than alignment of optimum temperatures or stabilities, rather the inflection points of enzymes in a metabolic pathways are aligned at the mean environmental temperature for the organism. This has the effect of coordinating relative enzyme rates and preventing metabolic disruption as temperature fluctuates. Also relevant here is that the response of metabolic pathways in general is not determined solely by a single enzyme. Prentice et al. show that, in general, the temperature-dependent properties of each enzyme in the pathway is important in determining the temperature dependence of the whole pathway.

   It is certainly important to understand what molecular features determine the temperature dependence of enzyme activity and its relationship to stability. Some previous proposals are mentioned in the manuscript. One important factor at the molecular level, mentioned by the authors, is work of Åqvist, Brandsval and coworkers, who have convincingly shown that activation entropy and enthalpy differ significantly between psychrophilic enzymes and their mesophilic and thermophilic counterparts. For small soluble enzymes, this is particularly due to changes at the enzyme surface, which may also affect stability. As mentioned by the authors, there have been many proposals over the years that suggest a relationship between stability and activity, though there is not a simple general relationship. Also directly relevant for the discussion here is what factors limit enzyme activity as temperature increases. The traditional view is that loss of activity is due to protein unfolding at high temperatures (the poor correlation of stability with growth temperatures found here indicates that this cannot be a general explanation). There is increasing evidence that this simple picture is wrong (see e.g. Daniel & Danson. (2010) Trends in Biochemical Sciences 35, 584). This behavior may be accounted for by conformational (e.g. two state) effects as proposed by Danson et al, distinct from the 'flexibility' proposals mentioned in the supporting information here. The introduction of the manuscript here states that "reaction rates are reduced at lower temperatures" , which might naively seem obvious but actually is not universally true, many reactions do not display simple Arrhenius-type behavior (see e.g. Kohen and Truhlar PNAS 2001 98 848). Many enzymes show a temperature of optimum activity, i.e. activity drops above the optimum temperature but before unfolding occurs. As the authors note, Arcus et al. show that this can be accounted for by an activation heat capacity, significantly larger in psychrophiles. Signatures of this behavior are apparent at the large scale (e.g. Schipper et al Global Change Biol. 2014 20 3578; Alster et al (2016) Front. Microbiol. 7:1821) and it appears to be generally important.

   Read the original source
7. 

   Version published to 10.1101/2021.08.25.457639v1 on bioRxiv

   Aug 25, 2021