Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase

Abstract

To efficiently catalyze a chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex, the chemical reaction and product release. These distinct demands could be satisfied via fluctuation between different conformational substates (CSs) with unique configurations and catalytic properties. However, there is debate as to how these rapid conformational changes, or dynamics, exactly affect catalysis. As a model system, we have studied bacterial phosphotriesterase (PTE), which catalyzes the hydrolysis of the pesticide paraoxon at rates limited by a physical barrier-either substrate diffusion or conformational change. The mechanism of paraoxon hydrolysis is understood in detail and is based on a single, dominant, enzyme conformation. However, the other aspects of substrate turnover (substrate binding and product release), although possibly rate-limiting, have received relatively little attention. This work identifies ''open'' and ''closed'' CSs in PTE and dominant structural transition in the enzyme that links them. The closed state is optimally preorganized for paraoxon hydrolysis, but seems to block access to/from the active site. In contrast, the open CS enables access to the active site but is poorly organized for hydrolysis. Analysis of the structural and kinetic effects of mutations distant from the active site suggests that remote mutations affect the turnover rate by altering the conformational landscape.dynamics ͉ enzyme catalysis ͉ evolution ͉ conformational fluctuation A catalyst is defined as a molecule that increases the rate of a chemical reaction by providing an alternative pathway of lower activation energy. A second, sometimes overlooked, requirement of a catalyst is that it is not consumed during the reaction, i.e., it must turn over multiple reactions. With reported rate enhancements of up to 10 17 (1) and turnover rates reaching 10 4 s Ϫ1 (2), enzymes are truly extraordinary catalysts. In addition to efficient rate enhancement of the chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex and product release. So, how does a single protein sequence satisfy these different demands?Our understanding of the mechanisms by which the active sites of enzymes lower the activation energy for various chemical reactions has developed steadily in the past decades and it is clear that the specific organization of chemical groups within the active sites of enzymes provides an electrostatic environment that is markedly different to the conditions in which the uncatalyzed reaction occurs, and results in remarkable rate enhancements (3). There have also been a number of studies showing that transitions between conformational substates (CSs) (4, 5) on a variable energy landscape (6, 7) are an integral part of the full catalytic cycles, or substrate turnover, in many enzymes (7-12). Despite much progress, there is some debate surrounding the exact role that such transitions, or dynamics, play in enzymatic catalysis (7, 13). The deb...

Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme

“…We also solved a structure of R18 with bound cacodylate, an analogue of the phosphodiester product of paraoxon hydrolysis, (1.50 Å resolution), and a structure of R18 in the absence of any ligand (1.85 Å). The binuclear metal ion centre of PTE at the active site (a-and b-sites) catalyses hydrolysis by stabilizing both the hydroxide nucleophile and the developing negative charge on the TS 23,28,32,33 . The metal ion centre also involves the carboxylation of a lysine residue 34 .…”

Reactibodies generated by kinetic selection couple chemical reactivity with favorable protein dynamics

“…Recently, the question of how enzymes harness protein dynamics for catalytic efficiency has been revisited through structural and computational approaches (1,40). Coupled conformational substates optimized to admit or release substrates or organize the active site for catalysis have been described (41).…”

Enzymatic Detoxication, Conformational Selection, and the Role of Molten Globule Active Sites

“…For substrate-specific enzymes and their cognate substrate, the breadth of the free enzyme conformational ensemble and substrate concentration determine whether the enzymes utilize conformational selection of pre-existing states to bind substrate, or the "classic" device of induced fit, in which substrate drives formation of a conformation that is otherwise unpopulated. Examples of both are abundant (5)(6)(7)(8), although conformational selection is more difficult to detect experimentally (9). However, the specific advantages and disadvantages of conformational selection and induced fit are not defined well.…”