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The Moderately Efficient Enzyme

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The Moderately Efficient Enzyme CURRENT TOPIC pubs.acs.org/biochemistry The Moderately Efficient Enzyme: Evolutionary and Physicochemical Trends Shaping Enzyme Parameters Arren Bar-Even,† Elad Noor,† Yonatan Savir,‡ Wolfram Liebermeister,† Dan Davidi,† Dan S. Tawfik,§ and Ron Milo*,† † ‡ § Department of Plant Sciences, Department of Physics of Complex Systems, and Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel bS Supporting Information ABSTRACT: The kinetic parameters of enzymes are key to understanding the rate and specificity of most biological processes. Although specific trends are frequently studied for individual enzymes, global trends are rarely addressed. We performed an analysis of kcat and KM values of several thousand enzymes collected from the “ ” ∼ À1 literature. We found that the average enzyme exhibits a kcat of 10 s and a kcat/ ∼ 5 À1 À1 ff KM of 10 s M , much below the di usion limit and the characteristic textbook portrayal of kinetically superior enzymes. Why do most enzymes exhibit moderate catalytic efficiencies? Maximal rates may not evolve in cases where weaker selection pressures are expected. We find, for example, that enzymes operating in secondary metabolism are, on average, ∼30-fold slower than those of central metabolism. We also find indications that the physicochemical properties of substrates affect the kinetic parameters. Specifically, low molecular mass and hydrophobicity appear to limit KM optimization. In accordance, substitution with phosphate, CoA, or other fi large modi ers considerably lowers the KM values of enzymes utilizing the substituted substrates. It therefore appears that both evolutionary selection pressures and physicochemical constraints shape the kinetic parameters of enzymes. It also seems likely that the catalytic efficiency of some enzymes toward their natural substrates could be increased in many cases by natural or laboratory evolution. À atural selection shapes the regulation of metabolic enzymes than 106À107 s 1.6,17 Furthermore, the apparent second-order Nenforcing precise spatial and temporal control. It also plays a rate for a diffusion-limited enzyme-catalyzed reaction with a central role in the evolution of enzymatic kinetic parameters, single low-molecular mass substrate (kcat/KM) cannot exceed ∼ 8À 9 À1 À1 18,19 including the turnover number (kcat) and the apparent binding 10 10 s M . The activation energy of the reaction, fl constants of substrates (KM), as both these parameters, together as re ected in the uncatalyzed rate, also comprises a barrier: the with enzyme concentrations, affect metabolism and growth rates. enzymatic acceleration of an extremely slow reaction, even by The literature usually highlights kinetically supreme enzymes many orders of magnitude, may still result in a relatively slow 5 such as triosephosphate isomerase, commonly termed a “perfect catalyzed rate. The overall thermodynamics of a reaction adds 20 enzyme”.1,2 What, however, are the kinetic parameters of the further limits. The Haldane relationship states a dependency “average enzyme”? Are most enzymes similarly “perfect”, and if between the kcat/KM of the forward (F) and backward (B) not, why? Here we describe a global view of enzyme parameters reactions, such that Keq =(kcat/KM)F/(kcat/KM)B, where Keq is ’ with the aim of highlighting the forces that shape the catalytic the reaction s equilibrium constant. Therefore, even when kcat/ ff efficiency of enzymes. KM in the favorable direction is di usion-limited, kcat/KM for the A large body of literature discusses the complex interplay unfavorable direction would be far lower than this limit. À among the various parameters of enzymatic catalysis,3 9 yet the Although there are no obvious limitations on KM, previous selective pressures that shaped these parameters remain largely studies have demonstrated that physicochemical properties of ligands and substrates correlate with their binding affinities unclear. While traditionally kcat/KM was thought to be an À 9 optimized quantity,4,8 10 other alternatives were proposed.1,11,12 (KS) toward receptors and enzymes. One example is the octanolÀwater partition coefficient (LogP), the ratio of concen- For example, KM values may have evolved to match physiological substrate concentrations,13 while a substrate-saturated enzyme is trations of a compound partitioning between a hydrophobic 8,14 expected to maximize kcat and to be insensitive to KM. There are also several known physicochemical constraints Received: February 14, 2011 that set boundaries to kinetic parameters.15,16 For example, Revised: April 11, 2011 theoretical limitations suggest that kcat is unlikely to be higher r XXXX American Chemical Society A dx.doi.org/10.1021/bi2002289 | Biochemistry XXXX, XXX, 000–000 Biochemistry CURRENT TOPIC a Table 1. Data Extracted for Our Analysis discussed in detail in the Supporting Information. This can be attributed to varying measurement conditions, such as pH, tem- no. of KM no. of kcat KM and perature, ionic strength, and the concentrations of metals and fi lter values values kcat cofactors, as well as differences in experimental methodologies, Brenda, all data 92516 36021 and inconsistencies between the values in the original papers and Brenda, removing mutants 76591 25157 the values entered in the Brenda database (Supporting In- Brenda, acting on KEGG natural substrates 31162 6530 formation). Further, most parameters in the literature are mea- sured in vitro, which can be considerably different from those unique reactions 5194 1942 1882 32,33 ff À measured in vivo. These di erences can be as great as 3 orders (enzyme substrate pairs) of magnitude.32 It will be worthwhile to revisit our results as more unique enzymes 2588 1176 1128 in vivo data become available. However, it is unlikely that such unique substrates 1612 834 804 differences will account for the systematic trends we observe. In a Each of the first three rows corresponds to the kinetic parameters addition, temperature significantly affects the kinetic parameters;34 downloaded from the Brenda website and filtered as described in the ° fl fi on average, kcat doubles from 25 to 37 C (Figure S2 of the Supporting Information. Brie y, in the rst step, parameters referring to Supporting Information). However, we tested such effects and mutant enzymes were discarded; in the second step, only parameters found that none of the trends described below results from a that refer to the natural substrate, as indicated by the KEGG database, were included. The last three rows correspond to the amount of unique systematic bias in assay temperatures (or pH values). parameters available after all the filtration steps. The last column gives Overall, the noisy nature of the data collected from the the number of reactions, enzymes, and substrates for which both K and literature limits the resolution of any global analysis and thus M ff kcat are available. limits the study to prominent di erences consistently observed in large samples. Moreover, certain subgroups of enzymes might display specific behavior not evident in other groups, thereby phase and a water phase. This indicator of hydrophobicity was masking global trends. Nevertheless, as discussed below, a global found to correlate with the binding energy of certain substrates analysis does uncover several general trends that underlie kinetic À toward their enzymes.21 24 In addition, previous studies found a parameters. correlation between the molecular mass of ligands and their receptor binding affinities.25,26 However, while binding of ligands ’ THE AVERAGE ENZYME IS FAR FROM KINETIC to receptors is a relatively simple process, the KM is a macroscopic value affected by microscopic rates that relate to substrate PERFECTION binding but also to enzymatic catalysis. The median turnover number for the entire data set of À Besides the physicochemical considerations, it was also sug- enzymes and natural substrates is ∼10 s 1 (Figure 1A), where ffi ∼ À À1 gested that the evolution of enzyme e ciency is dependent on most kcat values ( 60%) are in the range of 1 100 s . These the enzyme’s function within the organism and specific metabolic rates are orders of magnitude slower than the textbook examples context.1 Several cases were found to support this suggestion of fast enzymes [e.g., carbonic anhydrase (Figure 1A)], let alone À (e.g., ref 27). On the other hand, it appears that for many compared to the theoretical limit of 106À107 s 1.6,17 The fi ff ∼ 5 À1 À1 enzymes, signi cant reductions in rate have no e ect, and only median kcat/KM is 10 M s (Figure 1B), where most ffi ∼ 3À 6 À1 À1 relatively large reductions in catalytic e ciency hamper organis- kcat/KM values ( 60%) lie in the range of 10 10 M s , mal fitness (ref 28 and references cited therein). Thus, although it orders of magnitude below the diffusion limit. Although pooling is presumed that both evolutionary selection pressures and all enzymes to one distribution is conceptually problematic, this physicochemical constrains affect enzymes, the effects of these provides a unique perspective for a comparison of enzymes. For two factors on enzyme kinetic parameters are currently unclear. example, Rubisco, the central carbon fixation enzyme, is usually Here we present a global analysis of the kinetic parameters of considered to be a slow and inefficient enzyme.35 However, enzymes. We aim to highlight global factors shaping kinetic Figure 1 suggests that it actually possesses rather average kinetic parameters of enzymes rather than to discuss specific case parameters. studies. To allow such a global analysis, we have mined the When considering the maximal kcat/KM of each enzyme, the 29 30 ∼ Â 5 À1 À1 Brenda and KEGG databases. The Brenda database contains median kcat/KM is only 5-fold higher ( 5 10 M s ), the kinetic parameters for thousands of enzymes and substrates suggesting that constraints imposed by the Haldane relationship, collected from the literature. Using the KEGG database, we and the multisubstrate reactions of enzymes, only partially distinguished the parameters for the natural substrates of these explain why many enzymes operate far from the theoretical enzymes from those of non-native, promiscuous substrates kinetic limits.
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