aReproduced from O'Brien and Herschlag (1999) with permission of the authors and publisher.

aReproduced from O'Brien and Herschlag (1999) with permission of the authors and publisher.

TABLE 2.5 Enzymes with Promiscuous Catalysis of a Reaction that is also Catalyzed by Evolutionarily Related Enzymes3 Enzyme

Adenylate kinase Alkaline phosphatase

Arylsulfatase A Aspartate aminotransferase

D-Glucarate dehydratase O-Succinylbenzoate synthase Pyruvate oxidase Threonine synthase

Primary Activity

Phosphoryl transfer Phosphomonoester hydrolysis

Sulfate ester hydrolysis Amino group transfer

Dehydration Esterification Pyruvate oxidation Phosphate elimination

Promiscuous Activity

Sulfuryl transfer

Sulfate ester hydrolysis

Phosphodiester hydrolysis

Cyclic phosphodiester hydrolysis




N-Acylamino acid racemization Acetohydroxy acid synthesis Dehydration aReproduced from O'Brien and Herschlag (1999) with permission of the authors and publisher.

exergonic Mn -dependent dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1 -R-carboxylate (SHCHC) to 4-(2'-carboxylphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB) in the menaquinone biosynthetic pathway. This enzyme was first identified as an N-acylamino acid racemase (NAAAR), with optimal substrates being enantiomers of N-acetyl methionine. Palmer et al. (1999) subsequently discovered that the protein is a much better catalyst of the

OSB synthase reaction, with a 2.5 x 105 M"1 s value for

kcat/Km, for dehydration that greatly exceeds the 3.1 x 10 M"1 s"1 value for 1,1-proton transfer using the enantiomers of N-acetylmethionine. Taylor-Ringia et al. (2004) reported that the efficiency of the promiscuous NAAAR reaction is further enhanced with alternate substrates having structures mimicking the SHCHC substrate for the OSB synthase reaction; for example, the value of 2.0 x 105 M"1 s"1 for kcat/Km for enantiomers of N-succinyl phenylglycine compares to that for the OSB synthase reaction. The mechanisms of the NAAAR and OSB synthase reactions have been explored using mutants of Lys-163 and Lys-263 (Lys-163-Ala/Arg/Ser and Lys-263-Ala/Arg/Ser), the putative acid/base catalysts identified by sequence alignments with other OSBSs, including the structurally characterized OSB synthase from Escherichia coli (E. coli). Although none of the mutants display detectable OSB syn-thase or NAAAR activities, Taylor-Ringia et al. (2004) found that Lys-163-Arg and Lys-163-Ser catalyze stereo-specific exchange of the R-hydrogen of N-succinyl-(S)-phenylglycine with solvent hydrogen, and Lys-263-Arg and Lys-263 catalyze the stereospecific exchange of the R-hydrogen of N-succinyl-(R)-phenylglycine, a finding that is consistent with the formation of an Mn2+ stabilized eno-late anion intermediate. The rates of the exchange reactions catalyzed by the wild-type enzyme exceed those for race-mization. The discovery that this enzyme catalyzes two different reactions, each involving a stabilized enediolate anion intermediate, supports the hypothesis that evolution of function in the enolase superfamily proceeds by pathways involving functional promiscuity.

Vick, Schmidt and Gerlt (2005) later suggested that the natural pathway for enzyme evolution is apt to involve: (a) incremental increases in the level of the new reaction that would provide a selective advantage; and (b) an accompanying loss of the old reaction catalyzed by the progenitor. In an effort to better understand the molecular processes of divergent evolution, Schmidt et al. (2003) prepared the Asp-297 Gly mutant of E. coli L-Ala-D/L-Glu epimerase so that it could bind an OSB synthase substrate and thereby acquire OSB synthase activity. The epimerase progenitor did not catalyze the OSB synthase reaction, but the Asp-297-Gly mutant catalyzed a low level of the OSB synthase reaction (kcat = 0.013 s_1; Km = 1.8 mM; kcat/ Km = 7.4 M_1-s_1) that was sufficient to permit anaerobic growth by an OSB synthase-deficient strain of E. coli; the level of the progenitors natural epimerase reaction was significantly diminished. Using random muta-genesis and an anaerobic metabolic selection, they identified the Ile-19-Phe substitution as an additional mutation that enhances both growth of the OSB synthase-deficient strain and the kinetic constants for the OSB synthase reaction (kcat = 0.031 s-1; Km = 0.34 mM; kcat/ Km = 90 M^-s-1). Several other substitutions for Ile-19 also enhanced the level of the OSB synthase reaction. All of the substitutions substantially decreased the level of the epimerase reaction from that possessed by the Asp-297-Gly progenitor. The changes in the kinetic constants for both the OSB synthase and L-Ala-D/L-Glu epimerase reactions are attributed to a readjustment of substrate specificity so that the substrate for the OSB synthase reaction is more productively presented to the conserved acid/base catalysts in the active site. These observations support their hypothesis that evolution of new functions in the enolase superfamily can occur simply by changes in specificity-determining residues.

A highly intriguing example of catalytic promiscuity is the discovery by Yang and Metcalf (2004) that bacterial alkaline phosphatase BAP (Reaction: Phosphomonoester +

H2O # Alcohol + H2PO41 ) from E. coli is also phos-

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