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Mechanistic investigations of mandelate-racemase and muconate lactonizing enzyme I

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Title: Mechanistic investigations of mandelate-racemase and muconate lactonizing enzyme I
Author(s): Barrett, William C.
Doctoral Committee Chair(s): Gerlt, John A.
Department / Program: Biochemistry
Discipline: Biochemistry
Degree Granting Institution: University of Illinois at Urbana-Champaign
Degree: Ph.D.
Genre: Dissertation
Subject(s): Chemistry, Biochemistry
Abstract: Mandelate racemase (MR) and muconate lactonizing enzyme I(MLE) are found in the bacterium Pseudomonas putida. MR catalyzes the 1,1-proton transfer reaction equilibrating (R)- and (S)-mandelates. MLE catalyzes a reversible cycloisomerization of cis,cis-muconate and muconolactone. Both enzymes are characterized by the similar mechanistic feature of abstracting a relatively nonacidic $\alpha$-proton of a carboxylic acid. Superpositioning the two structures indicates nearly 60% of the $\alpha$-carbons are within 1.3 A of a structurally similar residue. These enzymes are believed to be divergently related.MR contains two active site bases, His297, the (R)-specific base, and Lys166, the (S)-specific base. MR contains a His297-Asp270 pair, that functions as a catalytic dyad. Asp270 was mutagenized to asparagine and k$\rm\sb{cat}$ of the reaction was decreased by 4 orders of magnitude and the pK$\rm\sb{a}$ of His297 perturbed from wild type MR. Asp270 is responsible for maintaining the proper pK$\rm\sb{a}$ value of His297.MLE contains one proposed active site acid/base, Lys169, the homolog to Lys166 in MR. Removal of the acid/base group of Lys169 resulted in a total loss of activity, however, with a general acid/base group, e.g., K169R, the k$\rm\sb{cat}$ decreased 10$\sp4$ from that of wild type MLE. Lys273 of MLE occupies a similar position in the X-ray structure to His297 and Asp270 of MR. Lys273 is believed to be necessary maintaining the hydrogen bond network within the active site of MLE. Lys273 was mutated and shown to be unnecessary for catalysis since K273A was active, however it is necessary for optimal catalysis as noted by a 4 orders of magnitude decrease in k$\rm\sb{cat}$ and 4-fold increase in K$\rm\sb{m}$.MR and MLE have an electrophilic catalyst, Glu317 and Glu327, to stabilize the intermediate. $\sp{13}$C-NMR studies have shown that the enolic intermediate in MR does not accumulate to an appreciable extent as predicted by the Gerlt-Gassman proposal for enzyme catalysis. Glu327 in MLE was mutated to E327Q and shown to form product at a rate 10$\sp4$-less than that of wild type MLE. The active sites of MR and MLE have many other features in common, but a combination of effects most likely prevents the catalysis of another reaction.
Issue Date: 1996
Type: Text
Language: English
URI: http://hdl.handle.net/2142/19530
ISBN: 9780591197389
Rights Information: Copyright 1996 Barrett, William C.
Date Available in IDEALS: 2011-05-07
Identifier in Online Catalog: AAI9712197
OCLC Identifier: (UMI)AAI9712197
 

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