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Enzyme Promiscuity: a Mechanistic and Evolutionary Perspective ANRV413-BI79-17 ARI 3 May 2010 8:37 Enzyme Promiscuity: A Mechanistic and Evolutionary Perspective Olga Khersonsky and Dan S. Tawfik Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel; email: tawfi[email protected] Annu. Rev. Biochem. 2010. 79:471–505 Key Words First published online as a Review in Advance on enzyme mechanism, protein evolution, superfamilies by California Institute of Technology on 12/14/10. For personal use only. March 17, 2010 Annu. Rev. Biochem. 2010.79:471-505. Downloaded from www.annualreviews.org The Annual Review of Biochemistry is online at Abstract biochem.annualreviews.org Many, if not most, enzymes can promiscuously catalyze reactions, or This article’s doi: act on substrates, other than those for which they evolved. Here, we 10.1146/annurev-biochem-030409-143718 discuss the structural, mechanistic, and evolutionary implications of Copyright c 2010 by Annual Reviews. this manifestation of infidelity of molecular recognition. We define All rights reserved promiscuity and related phenomena and also address their generality 0066-4154/10/0707-0471$20.00 and physiological implications. We discuss the mechanistic enzymology of promiscuity—how enzymes, which generally exert exquisite speci- ficity, catalyze other, and sometimes barely related, reactions. Finally, we address the hypothesis that promiscuous enzymatic activities serve as evolutionary starting points and highlight the unique evolutionary features of promiscuous enzyme functions. 471 ANRV413-BI79-17 ARI 3 May 2010 8:37 many enzymes are capable of catalyzing other Contents reactions and/or transforming other substrates, in addition to the ones for which they are phys- INTRODUCTION .................. 472 iologically specialized, or evolved, is definitely DEFINING AND QUANTIFYING not new. Early examples of enzyme promis- PROMISCUITY .................. 473 cuity include pyruvate decarboxylase (1), car- PROMISCUITY: RULE bonic anhydrase (2), pepsin (3), chymotrypsin OR EXCEPTION?................ 473 (4), and L-asparaginase (5). Nonetheless, the Specificity Is Context Dependent . 474 notion of “one enzyme—one substrate—one Regulation and Masking reaction” dominated, and still dominates the of Promiscuity .................. 474 textbooks, and until recently, the wider impli- Promiscuity within Living Cells .... 475 cations of the “darker” side of enzyme promis- The “Flexible Metabolome” ........ 475 cuity were largely ignored. MECHANISTIC ASPECTS OF The idea of nature as an opportunistic PROMISCUITY .................. 476 modifier of preexisting suboptimal functions Specificity and Promiscuity Coincide is also relatively old and has been formulated within the Same Active Site...... 476 by Jacob in his classical note “Evolution and Enzyme Mechanisms Analyzed by Tinkering” (6). The first direct connection Studying Promiscuous between promiscuity and protein evolution was Functions ...................... 480 made, to our knowledge, in 1976 by Jensen (7). Kinetic Parameters for Native versus Jensen boldly forwarded the hypothesis that, Promiscuous Functions ......... 480 unlike modern enzymes that tend to specialize PROMISCUITY AND in one substrate and reaction, the primordial, DIVERGENCE OF ENZYME ancient enzymes possessed very broad specifici- SUPERFAMILIES ................ 480 ties. Thus, relatively few rudimentary enzymes EVOLUTIONARY ASPECTS acted on multiple substrates to afford a wider OF PROMISCUITY .............. 485 range of metabolic capabilites. Divergence of Evolvability of Promiscuous specialized enzymes, via duplication, mutation, Functions: The Three Basic and selection, led to the current diversity of Postulates ...................... 486 enzymes and to increased metabolic efficiency. Promiscuous Functions Can Provide During the past decade, protein, and es- an Immediate Advantage ........ 486 pecially enzyme, promiscuity received con- Negative Trade-offs and the siderable attention. Reviews by O’Brien & Evolvability of Promiscuous Herschlag (8) and Copley (9) were the first to by California Institute of Technology on 12/14/10. For personal use only. Functions ...................... 486 highlight the potential mechanistic and evolu- Something for Nothing: For How Annu. Rev. Biochem. 2010.79:471-505. Downloaded from www.annualreviews.org tionary implications of promiscuity from an en- Long? .......................... 493 zymologist’s point of view. More recent reviews Exceptions to Weak Negative focused on practical implications of promiscu- Trade-offs ...................... 494 ity (1, 10–12), on promiscuity and divergence Stability Trade-offs................. 495 in specific enzyme families (13–16), on mech- Promiscuity and Mechanisms for anistic aspects of promiscuity (1, 17), and on Divergence of New Gene promiscuity in the context of protein evolution Functions ...................... 495 and design (17, 18). Here, we focus on the structural and mechanistic aspects of promiscuity as well as INTRODUCTION its role in the evolution of new functions. New Enzymes are traditionally referred to as re- enzymes have constantly emerged throughout markably specific catalysts. Yet the notion that the natural history of this planet. Enzymes that 472 Khersonsky · Tawfik ANRV413-BI79-17 ARI 3 May 2010 8:37 degrade synthetic chemicals introduced to the Commission (EC) numbers (33). In enzymes biosystem during the last decades (19–24), en- exhibiting multispecificity, or substrate ambi- zymes associated with drug resistance (25–28), guity, EC numbers for the various substrates Promiscuity: and enzymes in plant secondary metabolism should be the same, or differ only by the fourth coincidental catalysis (29–31) provide vivid examples of how fast digit (which generally distinguishes between of reactions other than and efficient the evolution of new enzymatic enzymes of the same class). Catalytic promis- the reaction(s) for functions can be. Indeed, extensive research cuity generally correlates with cases in which which an enzyme since Jensen’s article provided ample evidence the EC numbers of the various substrates and evolved for the notion that promiscuity is a key factor in reactions catalyzed by the same enzyme differ Multispecific or the evolution of new protein functions. Here, in the second, or the third, digits (which re- broad-specificity enzymes: enzymes we attempt to summarize this accumulating fer to different classes of substrates) or even by performing the same knowledge and point out some open questions the first digit (which indicates a different reac- reaction on a whole in this emerging field of research. tion category). (For examples see Supplemen- range of substrates, tal Table 1. Follow the Supplemental Mate- usually with similar rial link from the Annual Reviews home page efficiency DEFINING AND QUANTIFYING at http://www.annualreviews.org.) Substrate ambiguity: PROMISCUITY the activity of enzymes Magnitude of promiscuity refers to the ki- with substrates whose The term enzyme promiscuity (8) is loosely ap- netic parameters for the promiscuous activity structure resembles plied and is used to describe a wide range of relative to the native one. Whereas most en- the native substrate fundamentally different phenomena. We, and zymes exhibit kcat/KM values in the order of several others (8, 9, 32), use promiscuity to only 105–108 M−1s−1 for their native substrates, the describe enzyme activities other than the activ- magnitude of promiscuous activities varies over ity for which an enzyme evolved and that are more orders of magnitude, in absolute terms not part of the organism’s physiology. Thus, and also relative to the native activity. Catalytic enzymes, such as glutathione S-transferases proficiency (kcat/KM /kuncat) and rate acceleration (GSTs) and cytochrome P450s (33), which a (kcat/kuncat) can provide a measure of the magni- priori evolved to transform a whole range of tude of catalytic effects exerted on native ver- substrates, are not promiscuous; they are mul- sus promiscuous substrates. In many cases, al- tispecific or broad-specificity enzymes. though the kcat/KM values for the promiscuous Degree of promiscuity refers to the level of substrates are very low, and hence might have specificity breach, namely, how diverse are the little physiological relevance, the rate accelera- promiscuous activities of a given enzyme (34), tions and catalytic proficiencies are impressively and how different are the native and promiscu- high (34, 36–38). ous functions. The degree of promiscuity can be by California Institute of Technology on 12/14/10. For personal use only. assessed by examining the type of bonds that are Annu. Rev. Biochem. 2010.79:471-505. Downloaded from www.annualreviews.org being formed or broken and by differences in PROMISCUITY: RULE the mechanism between the native and promis- OR EXCEPTION? cuous reactions (10). An “index of promiscu- Numerous examples for enzyme promiscuity ity,” which computes the degree of variability are currently known, but these are anecdo- between different substrates, has also been pro- tal and hardly provide an indication for the posed (35). However, this method assumes that scale of this phenomenon. Systematic, high- the same chemical transformation occurs on throughput screens for promiscuous enzymatic all substrates. As such, it is more suitable for activities are not a feasible option at present; the analysis of multispecific enzymes such as no single detection method is available that can GSTs (as originally demonstrated), rather than detect the whole range of different substrates
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